Venkatachalam/aging_reg_dataset · Datasets at Hugging Face

POU5F1

5460

protein coding

MSC

--

Aging

Accelerate

Cell proliferation assay//Flow cytometry//SA--gal activity assay

When treated with doxycycline, OCT4A but not control vector-expressing MSC lost their spindleshaped structure and appeared fl at or shrunken in their morphology.There was a 50% decrease in the percentage of GFP-positive cells when OCT4A expression was induced, which was noticeable at 8 days post-doxycycline treatment, whereas no signifi cant changes were observed in the control vector-expressing cells.Cell-cycle analysis of the transgene-expressing cells showed that there was a G 1 cell cycle arrest during exogenous OCT4A expression in MSC.

--

Human

HL

Others

22,746,537

Gene

0

1

0

1

0

1

0

NEK4

6787

protein coding

HFF,BJ,293T,IMR-90,WI-38

Foreskin

Aging

Accelerate

BrdU assay//Knockdown//RT-PCR//SA--gal activity assay

Long-term culture of BJ cells in which NEK4 expression is suppressed (BJ shN4) revealed an extension of the time to replicative senescence of 10 to 20 PD compared to BJ shGFP cells.At late passage (PD 55 to 60), BJ shN4 cells proliferated at nearly twice the rate of BJ shGFP cells .Suppression of NEK4 by shN4 no. 1 resulted in a decrease in p21 mRNA levels smaller than that observed upon expression of shN4 no. 2 (0.81- and 0.27-fold, respectively), but we note that the levels of p21 mRNA and protein were still reduced compared to those in BJ shGFP cells.

--

Human

HL

Others

22,851,694

Gene

0

1

0

1

0

1

0

1

0

ZMAT3

64393

protein coding

MCF-7

Lung

Aging

Prevent

Cell proliferation assay//Colony formation assay//Flow cytometry//Knockdown//SA--gal activity assay

MCF7 breast cancer cells, which were treated with an siRNA against Wig1 (Wig Si), exhibited decreased cell proliferation and clonogenic ability.Interestingly, Wig1 depletion induced typical senescence associated phenotypic changes: large and flattened morphology, positive senescence-associated ¦Â-galactosidase (SA-¦ÂGal) staining, and G1 cell-cycle arrest. No significant increase in the subG1 phase was observed.

p21

Downregulation

qRT-PCR//Western blot//Knockdown

Overexpression and depletion of Wig 1 downregulated and upregulated p21, respectively, at both the mRNA and protein levels.The qRT¨CPCR assay revealed that Wig1-wt and Wig1-mut1 reverted the p21 3' UTR reporter mRNA levels that were increased due to Wig1 depletion to levels similar to those of Con Si-transfected cells, whereas Flag¨CWig1- mut2 failed to rescue these reporter mRNA levels.

--

Human

L

Others

23,085,987

Gene

0

1

0

1

0

1

0

1

0

1

0

PROX1

5629

protein coding

HepG2,Hep3B,Mahlavu,SK-HEP-1

Tumor tissue

Hepatocellular carcinoma

Accelerate

BrdU assay//SA--gal activity assay//Western blot

We used SK-HEP-1 cells for overexpression study and found that Prox1 reduced proliferation (as indicated by BrdU incorporation) in a time-dependent manner with a 30% of inhibition was found at 72 h after Prox1 expression in these cells.we found that percentage of ¦Â-galactosidase-positive cells was significantly increased after Prox1 overexpression indicating an induction of senescence-like phenotype.we examined the expression of CDKI proteins in control and Prox1-overexpressing cells. Among these inhibitory proteins, only p53 was dramatically increased.

p53

Upregulation

Knockdown//SA-¦Â-gal activity assay//Western blot

We first demonstrated that increase of ¦Â-galactosidase-positive cells by Prox1 expression was totally abolished by knockdown of p53 by shRNA. Human telomerase reverse transcriptase (hTERT) and chemokine C-X-C motif ligand 1 (CXCL1) have been shown to be downregulated and upregulated separately by p53 during p53-mediated senescence. We found that Prox1 significantly repressed hTERT expression and this effect was abolished when p53 was inhibited by shRNA.

--

Human

HL

Others

23,291,986

Gene

0

1

0

1

0

1

0

SOX2

6657

protein coding

HCT116

Tumor tissue

Colorectal cancer

Accelerate

Cell proliferation assay//Flow cytometry//SA--gal activity assay//Western blot

Our cell proliferation and cell cycle analyses indicated that Sox2 infection into HCT116 cells suppressed proliferation by inducing accumulation of cells in G0/G1 and sub-G1 and reduction of S cell cycle phases compared with mock-infected cells. The attenuation of cell proliferation was associated with about 90% inhibition of anchorage-independent cancer growth in soft agar, a hallmark of cancer cell properties.We then examined senescence using an SA-?galactosidase assay and found that ectopic expression of Sox2 enhanced ?-galactosidase activity and protein level and suppressed Ki-67 protein level, a cell proliferation marker.

ATG10

Upregulation

Knockdown//Luciferase reporter assay//Western blot//RT-PCR//Immunochemical staining

Our cycledependent RT-PCR results demonstrated that the ATG10 mRNA level from Sox2-transfected cells was increased by about 2.5 fold compared with mock.Notably, Western blotting results and immunocytofluorescence against ATG10 indicated that ATG10 and LC3 protein levels were increased by overexpression of Sox2.We found that that deletion of putative Sox2 binding region significantly suppressed luciferase activity. Notably, ATG10 promoter activity was increased by overexpression of Sox2.

--

Human

HL

Others

23,451,179

Gene

0

1

0

1

0

1

0

1

0

YAP1

10413

protein coding

IMR-90

--

Aging

Prevent

BrdU assay//Flow cytometry//Knockdown//SA--gal activity assay

Compared with the control, silencing YAP significantly induced cellular senescence. SA-¦Â-gal staining and BrdUrd incorporation were used as readouts for senescence. Knockdown of YAP increases the expression of SA-¦Â-gal and inhibits cell DNA replication.Furthermore, YAP downregulation also resulted in the inhibition of cell proliferation in a population doubling assay.As expected, YAP knockdown significantly reduced the S-phase and increased the G1-phase in the IMR90 cells.

CDK6

--

qRT-PCR//Western blot//CHIP//Knockdown//SA-¦Â-gal activity assay

In our experiments, YAP or TEAD knockdown significantly inhibited CDK6 mRNA level and protein expression in IMR90 cells.Further ChIP experiments confirmed the binding of YAP to these 2 sites, whereas we used the already identified YAP¨CTEAD target CTGF as a positive control. Furthermore, overexpression of CDK6 can partially rescue the increased senescence induced by YAP knockdown.

TEAD//Rb-p53-p16

--//--

Knockdown//SA-¦Â-gal activity assay//BrdU assay//qRT-PCR

We found that knockdown of TEAD1/3/4 also induces an almost identical cellular senescent phenotype as YAP silencing. Further solidifying our hypothesis that YAP regulates cellular senescence in a TEAD-dependent manner, we found that the knockdown of boIn our SA-¦Â-gal staining experiments, silencing either p16 or p53 did not rescue the senescent phenotype caused by YAP knockdown th YAP and We also found that the individual knockdown of p53 or p16 could not rescue the YAP knockdown-induced senescent phenotype by conducting population doubling assay experiments. However, double knockdown of p53 and p16 partially rescued YAP knockdown-induced senescence , suggesting that YAP antagonized senescence through both the p53 and p16 pathways TEAD1/3/4 together did not lead to a further increased senescent phenotype, including SA-¦Â-gal staining and BrdUrd incorporation as well as PDL.

Human

L

Others

23,576,552

Gene

0

1

0

1

0

1

0

1

0

NOTCH3

4854

protein coding

BJ

--

Aging

Accelerate

Knockdown//SA--gal activity assay//Western blot

Downregulation of Notch3 in mid-passage cells resulted in a delayed onset of replicative senescence and an extended replicative lifespan,Downregulation of Notch3 by shRNA resulted in decreased p21 expression, suggesting that Notch3 is required for elevated p21 expression in senescent cells.

p21

Upregulation

Knockdown//SA-¦Â-gal activity assay//Western blot//Cell proliferation assay

Similar to BJ and WS1 fibroblasts, ectopic expression of NICD3 in LF1 cells induced senescence and p21 expression.However, the ability of Notch3 to induce senescence was significantly attenuated in p21- /- cells, as indicated by increased cell proliferation and decreased SA-¦Â-gal staining compared with the parental LF1 cells. These results suggest that Notch3-induced senescence is partially mediated by p21.

--

Human

HL

Others

23,610,446

Gene

0

1

0

1

0

1

0

PEBP1

5037

protein coding

hTERT-immortalized p21?/?,LF1,PC-3,HCT116

--

Aging

Accelerate

Knockdown//MTT assay//SA--gal activity assay//Western blot

Elimination of RKIP could increase ERK activation and suppress the cellular senescence. Conversely, RKIP overexpression inhibited ERK activation and induced the cellular senescence (monitored by SA-¦Â-Gal and DcR2 expression) in two kinds of p53-deficient cell lines.As we expected, overexpression of RKIP could suppress the cell proliferation, whereas RKIP knockdown could promote the cell proliferation.

--

Ras-Raf-ERK

Downregulation

Knockdown//SA-¦Â-gal activity assay//Western blot

Suppression of Ras-Raf-ERK pathway using Raf kinase inhibitor could induce the cellular senescence .Elimination of RKIP could increase ERK activation and suppress the cellular senescence. Conversely, RKIP overexpression inhibited ERK activation.

Human

L

Others

23,814,485

Gene

0

1

0

1

0

1

0

1

0

DPY30

84661

protein coding

IMR-90,NT2

--

Aging

Prevent

Cell proliferation assay//Colony formation assay//Growth curve assay//Knockdown//SA--gal activity assay//Western blot

The overall morphology of the DPY30 knockdown IMR90 cells changed, towards a more rounded and flattened phenotype with enlarged nuclei.SA-¦Â-galactosidase assays showed that 490% of the shDPY30 IMR90 cells expressed Activate SA-¦Â-galactosidase. Further, in proliferation assays, growth curves, and colony formation assays, shDPY30 IMR90 cells arrested and stopped proliferating.

ID

--

qRT-PCR//Western blot//Knockdown//SA-¦Â-gal activity assay

In ID1 and ID3 overexpressing cells, DPY30 knockdown reduced mRNA levels, but not proteins levels, of ID1 and ID3, the shDPY30 cells with ID1 or ID3 overexpression regained their fibroblast features and began to proliferate again, albeit not to the same degree as the IMR90 control cells, while the shDPY30 cells remained in a senescence-like state .

--

Human

HL

Others

23,872,946

Gene

0

1

0

1

0

1

0

1

0

1

0

1

0

ID4

3400

protein coding

DU 145

--

Prostate cancer

Accelerate

Immunostaining//SA--gal activity assay//Western blot

Enlarged morphology and accumulation of cytoplasmic aggregates were observed in DU145+Id4 cells as compared to DU145 cells,indicative of senescence.An increase in senescence associated beta-galactosidase (SA-¦Âgal) staining suggested that ectopic expression of Id4 in DU145 cells promotes senescence at a higher frequency than in un-transfected DU145 cells.Immunocytochemical analysis shown clearly indicate that Id4 up-regulates G1 cell- cycle regulators p21 and p2, as compared to DU145 cells. We also observed an increased p16 expression at protein and transcript levels in DU145+Id4 cells, compared to DU145 cells but its functional relevance in DU145 cells with respect to senescence remains obscure.

E2F1

Downregulation

Western blot

At 10 nm, a significant increase in senescence was not observed in DU145 cells. The basal (0 h) E2F1 expression was significantly lower in DU145+Id4 cells as compared to DU145 cells whereas E2F1 expression was almost undetectable in DU145+Id4 cells at subsequent time points.

--

Human

L

Others

24,122,992

Gene

0

1

0

1

0

1

0

RB1

5925

protein coding

Primary human bone cell

Bone

Retinoblastoma

Accelerate

Knockdown//SA--gal activity assay//qRT-PCR

Consistent with a role in senescence, radiation-induced expression of Il1b, Il6, Il8/Mip2, and Mcp1 was markedly attenuated in Rb1fl/fl mice relative to that in wild-type mice . Confirming these findings, ex vivo studies using 4 Gy IR also showed decreased SA-¦Â-Gal¨Cpositive staining (data not shown) and reduced protein expression of IL-6 and MCP-1 in calvaria from Rb1fl/fl mice compared with that in wild-type mice.

--

Human

HL

Others

24,231,354

Gene

0

1

0

1

0

1

0

HSPA5

3309

protein coding

A2780

Bone

Ovarian cancer

Prevent

Knockdown//SA--gal activity assay

Seven days after 3 ¦Ìg/ml cisplatin treatment, <20% of the A23187-treated A2780 cells showed positive SA-¦Â-gal staining, whereas cells treated with cisplatin only were almost all positively stained. To confirm the specific anti-senescence effect of GRP78, GRP78-siRNA was transfected into the A23187-treated A2780 cells. After 3 days, the treated cells were exposed to 3 ¦Ìg/ml cisplatin and incubated with complete medium. After 6 days,the cells exhibited 90% positive SA-¦Â-gal staining.

p53//CDC2

--//--

Knockdown//Western blot

The results showed that GRP78 protein levels were significantly decreased after GRP78-siRNA transfection in the A2780 cells. Accompanied by knockdown of GRP78, P53 levels were significantly increased and CDC2 levels were significantly decreased. There were no changes in the protein levels of P21 and p-CDC2. This suggests that P53 and CDC2 are involved in cisplatin-induced senescence of A2780 cells.

--

Human

L

Others

24,756,776

Gene

0

1

0

1

0

ERRFI1

54206

protein coding

WI-38

--

Retinoblastoma

Accelerate

SA--gal activity assay

Myc¨CMig-6 overexpression increased cell senescence as evidenced by the elevated number of the SA-?-gal positive, blue coloured cells compared to vector control transfected fibroblasts.

--

ERK//AKT

Activation//Activation

Western blot

The strong signal for phosphorylated EGFR (pEGFR Tyr1173) observed in early passage cells at 5 min of EGF stimulation indicated the quick and transient response . Late passage (P25) fibroblasts exhibited an attenuated acute response to EGF treatment as evidenced by the slightly decreased levels of Tyr1173 phosphorylated EGFR at 5 min.Downstream of the EGFR, ERK activity was sustained in late passage as compared to early passage cells, whereas AKT activity was massively down-regulated in late passage cells . Moreover, the expression level of p16INK4A, a typical senescence-associated protein, was generally elevated in late passage cells as compared to young fibroblasts.

Human

L

Others

24,815,188

Gene

0

1

0

LGALS3

404021

protein coding

AGS,MEF

Stomach

Retinoblastoma

Prevent

Cell proliferation assay//Flow cytometry//Knockdown//SA--gal activity assay

Treatment of AGS cells with 10 mM galectin-3 siRNA (Gal3 siRNA) for 2 days reduced cell proliferation by 50%.The depletion of galectin-3 significantly increased the number of G1 cell cycle-arrested cells and the sub-G1 populations, whereas co-depletion of galectin-3 and p27KIP1 produced equivalent proliferation to scRNA control group cell populations.Galectin-3 -/- MEFs showed growth retardation when compared with galectin-3 +/+ MEFs.Depletion of galectin-3 resulted in growth retardation and premature senescence in the human foreskin fibroblasts.

p27

--

Knockdown//Western blot//RT-PCR//SA-¦Â-gal activity assay//Cell proliferation assay

Galectin-3 depletion was accompanied by decreased Skp2 and increased p27KIP1 expression. Although the expression of p16INK4A and p21WAF1/CIP1 remained unchanged, p14ARF levels were found to decrease slightly.Galectin-3 depletion reduced Skp2 mRNA but increased p27KIP1 protein expression without a change in the mRNA expression. This suggests that galectin-3 regulates the level of p27KIP1 protein by Skp2-mediated ubiquitin activity.Upon galectin-3 depletion, both the cell types exhibited growth retardation and additional p27KIP1 ablation reversed this growth retardation .

--

Human

HL

Others

24,971,481

Gene

0

1

0

1

0

1

0

1

0

DHX9

1660

protein coding

MRC-5

--

Aging

Prevent

Flow cytometry//Growth curve assay//Knockdown//SA--gal activity assay

DHX9-suppressed cells stained positive for SA ¦Â-gal, a distinguishing marker of senescent cells.DHX9 shRNA-expressing cells show a significantly higher percentage of SA¦Â -gal-positive cells compared with MRC-5 controls (50¨C57% for DHX9 knockdown cells compared with 3% for shFLuc.1309-expressing cells). Measurements of the growth rates of DHX9-depleted MRC-5 cells indicated these to be at least 2-fold lower than control cells and similar to hRAS V12-expressing cells . Cell cycle analyses showed an increase in the percentage of cells in the G0/G1 phase and a decrease in the percentage of cells in the S and G2 phases 14 days after infection of DHX9 shRNAs.

--

p53

--

Knockdown//SA-¦Â-gal activity assay//Western blot

Loss of p53 completely abolished the SA-¦Â-gal staining and rescued the growth defect in DHX9 knockdown cells. In addition, loss of p53 eradicates the p21 and RB1 responses in the DHX9 knockdown cells, compared with the vector control .This demonstrates that p53 is essential for DHX9-induced senescence.

Human

HL

Others

24,990,949

Gene

0

1

0

1

0

1

0

1

0

HMGB1

3146

protein coding

A375,G361

Melanoma

Prevent

Cell proliferation assay//Flow cytometry//Knockdown//SA--gal activity assay

Knockdown of HMGB1 expression resulted in a marked reduction of cell proliferation and importantly, this effect appeared to be nicely correlated with the level of HMGB1 expression.Relative to the control cells, HMGB1- deficient cells showed a significant delay in cell cycle progression.We noticed that the HMGB1 stable knockdown cells appeared to be flatter in shape and larger in size than the matched control cells.This morphology change prompted us to ask whether HMGB1 depletion might induce cellular senescence. Measurement of senescent-associated ¦Â-galactosidase activity showed a marked increase of ¦Â-Gal-positive cells.

p21

--

Knockdown//SA-¦Â-gal activity assay//Flow cytometry

The result indicates that depletion of p21 expression almost completely abrogated G1 cell cycle arrest induced by HMGB1 deficiency.In addition,knockdown of p21 expression also abolished the senescent phenotype in HMGB1 deficient cells.

SP1

--

Luciferase reporter assay

Consistent with a Sp1-dependent and p53-independent mechanism of p21 regulation, mutation of Sp1 but not p53 binding sites abrogated p21 induction by HMGB1-deficience. Almost identical results were obtained in both A375 and SK-MEL-28 cell lines. These data together confirm a Sp1-depdent mechanism of p21 induction by HMGB1-depletion.

Human

HL

Others

25,051,367

Gene

0

1

0

1

0

1

0

1

0

SELENOH

280636

protein coding

MRC-5

--

Aging

Prevent

Cell proliferation assay//Knockdown//SA--gal activity assay

Moreover, the complete growth inhibition of SelH shRNA MRC5 cells maintained under 20% O2 for 5 weeks could be partially rescued when grown in a 3% O2 incubator, although SelH shRNA MRC-5 cells remained to proliferate poorly (~300-fold slower) as compared to scrambled shRNA MRC-5 cells.the percent SA-¦Â-gal positive cells 5 days after recovery were 17% and 70% in scrambled shRNA and SelH shRNA MRC-5 cells, respectively, in a 20% O2 incubator. When maintained under 3% O2, they dropped (p < 0.05) to 2% and 34 % in scrambled and SelH shRNA MRC-5 cells, respectively.

--

ATM-p53

--

Knockdown//SA-¦Â-gal activity assay//Proliferation assay

Treatment with Ku 60019 (5 ¦ÌM), a specific ATM kinase inhibitor (53), for 28 days alleviated the slow proliferation phenotype by 3- fold in SelH shRNA MRC-5 cells, but slightly suppressed the proliferation in scrambled shRNA MRC-5 cells.Analyses of SA-¦Âgal staining confirmed that the ATM kinase is required to senesce SelH shRNA cells (40% vs 5%) after being cultured for 28 days. SelH shRNA MRC-5 cells significantly accumulated (p < 0.05) additional H2AX and pATM Ser-1981 after 28 days in a 20% O2 incubator, but such induction was completely.

Human

L

Others

25,336,634

Gene

0

1

0

1

0

1

0

PRKCH

5583

protein coding

MCF-7

--

Aging

Accelerate

Cell proliferation assay//Colony formation assay//Knockdown//SA--gal activity assay//Western blot

In response to H2O2 or etoposide, PKC¦Ç-knockdown cells showed significantly and reproducibly lower number of SA-¦Â-galactosidase-stained cells compared with scrambled control cells.A lower number of proliferating cells were regrown out of senescent cultures of scrambled (sh scr 5-3) control cells compared with PKC¦Ç-knockdown (sh 3-3 and sh 2-2) cells in colony formation assays. Viability assays (Neutral Red) with additional PKC¦Ç-knockdown clones (sh 4-2 and sh 3-5) showed similar results (data not shown).p21Cip1 is required to induce senescence. Our results showed increased expression of p21Cip1 and p27Kip1 in PKC¦Çexpressing cells (sh scr 5-3), which was markedly reduced in PKC¦Ç-knockdown cells upon etoposide treatment.

--

Human

L

Others

25,412,309

Gene

0

1

0

1

0

1

0

1

0

1

0

PDCD10

11235

protein coding

IMR-90

--

Aging

Accelerate

BrdU assay//Knockdown//SA--gal activity assay//qRT-PCR

When CCM3 was inhibited in these cells,4-OHT could still increase the number of ¦ÃH2AX-positive cells,showing that H-ras was Activate in both control and CCM3-deficient cells. However, it did not inhibit DNA synthesis, nor did it induce a senescent morphology or senescence-associated ¦Â-galactosidase activity.

--

Human

HL

Others

25,655,101

Gene

0

1

0

1

0

1

0

1

0

P3H1

64175

protein coding

IMR-90

--

Aging

Prevent

Cell proliferation assay//Colony formation assay//Knockdown//SA--gal activity assay

The down-regulation of LEPRE1, LIMA1, MAGOHA, and MAGOHB caused a significant reduction in cell growth (p ¡Ü 0.05) at 4 days after transfection, when compared with a negative siRNA control. In parallel, we tested if knockdown of the nine genes also induces the SA-¦Â-gal activity.SiRNAmediated down-regulation of LEPRE1, LIMA1, and MAGOHA and MAGOHB robustly increased the number of SA-¦Â-gal-positive cells, compared to cells transfected with siRNA control.a clonogenic test performed on young IMR90 cells after the inhibition of LEPRE1, LIMA1, MAGOHA or MAGOHB by the corresponding siRNAs demonstrated that LEPRE1 or LIMA1 down-regulation strongly inhibited the formation of colonies, compared with a negative siRNA control, while MAGOHA or MAGOHB down-regulation hampered treated cells from resuming proliferation.

--

Human

L

Others

26,206,181

Gene

0

1

0

1

0

1

0

1

0

LIMA1

51474

protein coding

IMR-90

--

Aging

Prevent

Cell proliferation assay//Colony formation assay//Knockdown//SA--gal activity assay

The down-regulation of LEPRE1, LIMA1, MAGOHA, and MAGOHB caused a significant reduction in cell growth (p ¡Ü 0.05) at 4 days after transfection, when compared with a negative siRNA control. In parallel, we tested if knockdown of the nine genes also induces the SA-¦Â-gal activity. SiRNAmediated down-regulation of LEPRE1, LIMA1, and MAGOHA and MAGOHB robustly increased the number of SA-¦Â-gal-positive cells, compared to cells transfected with siRNA control.a clonogenic test performed on young IMR90 cells after the inhibition of LEPRE1, LIMA1, MAGOHA or MAGOHB by the corresponding siRNAs demonstrated that LEPRE1 or LIMA1 down-regulation strongly inhibited the formation of colonies, compared with a negative siRNA control, while MAGOHA or MAGOHB down-regulation hampered treated cells from resuming proliferation.

--

Human

L

Others

26,206,181

Gene

0

1

0

1

0

1

0

1

0

MAGOHA

4116

protein coding

IMR-90

--

Aging

Prevent

Cell proliferation assay//Colony formation assay//Knockdown//SA--gal activity assay

The down-regulation of LEPRE1, LIMA1, MAGOHA, and MAGOHB caused a significant reduction in cell growth (p ¡Ü 0.05) at 4 days after transfection, when compared with a negative siRNA control. In parallel, we tested if knockdown of the nine genes also induces the SA-¦Â-gal activity. SiRNAmediated down-regulation of LEPRE1, LIMA1, and MAGOHA and MAGOHB robustly increased the number of SA-¦Â-gal-positive cells, compared to cells transfected with siRNA control.a clonogenic test performed on young IMR90 cells after the inhibition of LEPRE1, LIMA1, MAGOHA or MAGOHB by the corresponding siRNAs demonstrated that LEPRE1 or LIMA1 down-regulation strongly inhibited the formation of colonies, compared with a negative siRNA control, while MAGOHA or MAGOHB down-regulation hampered treated cells from resuming proliferation .

--

Human

L

Others

26,206,181

Gene

0

1

0

1

0

1

0

1

0

MAGOHB

55110

protein coding

IMR-90

--

Aging

Prevent

Cell proliferation assay//Colony formation assay//Knockdown//SA--gal activity assay

The down-regulation of LEPRE1, LIMA1, MAGOHA, and MAGOHB caused a significant reduction in cell growth (p ¡Ü 0.05) at 4 days after transfection, when compared with a negative siRNA control. In parallel, we tested if knockdown of the nine genes also induces the SA-¦Â-gal activity. SiRNAmediated down-regulation of LEPRE1, LIMA1, and MAGOHA and MAGOHB robustly increased the number of SA-¦Â-gal-positive cells, compared to cells transfected with siRNA control.a clonogenic test performed on young IMR90 cells after the inhibition of LEPRE1, LIMA1, MAGOHA or MAGOHB by the corresponding siRNAs demonstrated that LEPRE1 or LIMA1 down-regulation strongly inhibited the formation of colonies, compared with a negative siRNA control, while MAGOHA or MAGOHB down-regulation hampered treated cells from resuming proliferation.

--

Human

L

Others

26,206,181

Gene

0

1

0

1

0

1

0

1

0

DUSP16

80824

protein coding

PLC-PRF-5

Human HCC sample,Tumor tissue

Aging

Prevent

BrdU assay//Flow cytometry//Knockdown//SA--gal activity assay//TUNEL assay

TUNEL and Propidium Iodide (PI) staining, respectively. TUNEL-positive signals could be detected in DNase I or chemotherapeutic drug doxorubicin treated cells, but not in DUSP16-silenced cells ,By contrast, we found that upon DUSP16 knockdown, the PLC/PRF/5 cells were blocked in the G1 phase, suggesting that these cells were unable to initiate DNA replication and maintained a growth-arrested state. We also noticed that DUSP16-silenced cells exhibited a lower BrdU incorporation rate when compared with control cells.In accordance with the impaired cell cycle transition, the cell cycle-related proteins CDK1, CDK2, CDK4 and cyclin E were markedly down-regulated in DUSP16-silenced cells. SA-¦Â-Gal activity is a widely used marker for measuring cellular senescence. Intriguingly, the percentage of SA-¦Â-Gal-positive cells significantly increased upon DUSP16 silencing.

--

p53//Rb

--//--

Knockdown//SA-¦Â-gal activity assay//Western blot//qRT-PCR

Upon DUSP16 knockdown, p53 downstream effectors were dramatically up-regulated .In addition, we found that upon DUSP16 knockdown, the increase of SA-¦Â-Gal-positive cells and the decrease of the number of viable cells were efficiently attenuated by shRNA-mediated p53 silencing .Interestingly, in PLC/PRF/5 cells, we identified that decreased levels of CDKs upon DUSP16 silencing led to reduced phosphorylation of Rb , suggesting that the activation of Rb is involved in DUSP16 silencing-induced senescence in p53 mutant liver cancer cells .In addition, in the HepG2 cell line that carries wild-type p53, our results showed increased phosphorylation of p53, as well as, reduced phosphorylation of Rb upon DUSP16 silencing ,suggesting that both p53 and Rb are likely downstream effectors of DUSP16 silencing-induced senescence in p53 wild-type liver cancer cells.

Human

HL

Others

26,381,291

Gene

0

1

0

1

0

1

0

1

0

1

0

GATA4

2626

protein coding

IMR-90,BJ

Brain

Aging

Accelerate

BrdU assay//SA--gal activity assay

Ectopic expression of GATA4 induced senescence in human foreskin fibroblasts and IMR-90 fibroblasts,as shown by increased senescence-associated ¦Â-galactosidase (SA-¦Â-Gal) activity and decreased 5-bromo-2¡ä-deoxyuridine (BrdU) incorporation.

TRAF3IP2//IL1A

--//--

Knockdown//Western blot//qRT-PCR

TRAF3IP2 depletion partially blocked GATA4 activation of NF-kB, as assessed by the expression of SASP genes.Furthermore, ectopic expression of TRAF3IP2 partially rescued the reduced SASP caused by GATA4 depletion in IR-induced senescent cells.Depletion of IL1A reduced GATA4 activation of NF-kB, as assessed by the expression of SASP genes. Furthermore, activation of SASP-associated genes was transmitted to cells lacking GATA4 induction by conditioned medium from GATA4-induced cells.Thus, GATA4 appears to act, at least in part, through TRAF3IP2 and IL1A to activate NF-kB in activating the SASP.

NF-¦ÊB

Activation

Western blot

GATA4 expression triggered NF-kB activation, and GATA4 depletion inhibited NF-kB activation during senescence .

Human

HL

Others

26,404,840

Gene

0

1

0

1

0

NANOG

79923

protein coding

NIH-3T3 MEF,Mouse Fibroblast cell

--

Aging

Prevent

Cell proliferation assay//SA--gal activity assay

Nanog-TAT caused a strongly increased proliferation, yielding about four-fold more cells within 10 days as compared to the control.Approximately 6% of MP-hADFs cultured under normal conditions for 3 passages stained positive for SA-¦Â-gal. In contrast, no SA-¦Â-gal activity was detectable in MPhADFs cultured in the presence of Nanog-TAT.

p27

Downregulation

RT-PCR//Western blot

After 5 h of Nanog-TAT treatment, the RNA expression level of p27KIP1 was slightly reduced, to ¡«90% compared to fibroblasts cultured in control medium. After 8 h of Nanog-TAT application, the expression of p27KIP1 was diminished to¡«70%. After 21 h of Nanog transduction, we detected only ¡«25% p27KIP1 transcript as compared to cells incubated with control medium.Quantification demonstrates that 5 h of Nanog transduction is sufficient to reduce the p27KIP1 protein level to about half compared to control; 24 h of incubation with Nanog-TAT yielded a further decline of p27KIP1 to ¡«40% compared to control.

--

Human

L

Others

26,795,560

Gene

0

1

0

1

0

HRAS

3265

protein coding

MEF

Embryo

Lung cancer

Accelerate

Cell morphological analysis

In the case of overexpressed oncogenic H-Ras, cells were analyzed 6 days post-infection when, as expected, cells had a clear senescent morphology.

--

Human

L

genomic instability

19,421,407

Gene

0

1

0

TFDP1

7027

protein coding

TIG-3

--

Aging

Prevent

Knockdown//SA--gal activity assay//SAHF

Furthermore, several features of cellular senescence, such as a substantial increase in senescence-associated¦Â-galactosidase (SA-¦Â-gal) activity and senescence-associated heterochromatic foci (SAHF) were observed in DP1 knock-down cells,but not in cells expressing flag-tagged DP1 that is resistant to RNAi (unpublished data).

--

Human

L

delay aging

15,716,376

Gene

0

1

0

1

0

1

0

MIR200A

406983

ncRNA

Keratinocyte

--

Aging

Accelerate

Western blot//qRT-PCR

Altogether, these findings point miR-200a as key player in primary human keratinocyte aging since its overexpression reduces oxidative DNA repair activity and may induce cell cycle arrest via p16 up-regulation and fuels chronic inflammation via NLRP3 activation.

OGG1-2a//NRLP3//IL-1¦Â//Bmi-1//p16

Downregulation//Upregulation//Upregulation//Downregulation//Upregulation

Luciferase reporter assay//qRT-PCR//Western blot

HEK 293 cells have been cotransfected with a construct containing the OGG1-2a 3¡ä-UTR downstream of a luciferase open reading frame and either with miR-200a or a miR-scramble. Relative luciferase activity was significantly down-regulated (~29%) upon miR-200a overexpression.As expected, following miR-200a expression ,a significant decrease of OGG1-2a expression was observed in both cell types.Of note, miR-200a overexpression induced also a significant increase of NRLP3, caspase 1, and IL-1¦Â expression mainly in fibroblasts.Following miR-200a expression, Bmi-1 expression significantly decreased at both mRNA and protein levels in HEK 293 cells and primary human fibroblasts. Simultaneously, a significant increase of the senescence mediator p16 was observed .

--

Human

HL

cellular senescence

29,765,508

Gene

0

1

0

1

0

LINC00673

100499467

ncRNA

A549,IMR-90

--

Lung cancer

Prevent

Cell morphological analysis//Flow cytometry//Knockdown//SA--Gal activity assay

LINC00673 knockdown caused a change of cellular morphology, namely increased cell size and adaptation of a flat cell morphology in both A549 lung cancer cells and IMR-9 normal lung fibroblast cells.The reduction of LINC00673 levels in A549 cells was accompanied by a prominent increase of cells in G0/G1-phases and a concomitant decrease of cells in S-phase .We confirmed a strong increase of SA-¦Â-Gal positive A549 and IMR-9 cells 4 days after LINC00673 knockdown.

--

p53

--

SA-¦Â-Gal activity assay

SiPOOL-mediated depletion of p53 was sufficient to overcome senescence triggered by LINC00673 depletion in A549 cells.

Human

HL

cellular senescence

30,499,379

Gene

0

1

0

1

0

1

0

1

0

CircLARP4

--

ncRNA

MHCC97L,HCCLM3

--

Hepatocellular carcinoma

Accelerate

Flow cytometry//Knockdown//SA--Gal activity assay

We performed flow cytometry to analyze the cell cycle distribution.CircLARP4 overexpression caused G1/S cell cycle arrest in MHCC97L, whereas knockdown of circLAPR4 exhibited the opposite effects in HCCLM3 cells.To verify our hypothesis, we performed SA-¦Â-gal staining to determine the cellular senescence. Data showed that the percentage of SA-¦Â-gal-positive cells in circLARP4-overexpressed MHCC97L cells was significantly increased.

--

miR-761-RUNX3-p53-p21

--

Knockdown//Western blot//FISH//RIP//Luciferase reporter assay//Immunohistochemistry

Western blotting showed that the expression levels of p53 and p21 were upregulated in circLARP4-overexpressed MHCC97L cells and downregulated in sh-circLARP4 HCCLM3 cells.MiR-761 expression was remarkably decreased in circLARP4-overexpressed MHCC97L cells, while miR-761 expression was elevated in sh-circLARP4 HCCLM3 cells . FISH analysis in HCC cells showed that circLARP4 was co-localized with miR-761 in cytoplasm. Given that high degree of AGO2 occupancy in circLARP4 has been implicated previously,16 we performed RIP for AGO2 in HCC cells and showed higher expression levels of circLARP4 and miR-761 in AGO2 pellet compared to those in the control group . Subsequent luciferase reporter assays demonstrated that miR-761 was a direct target of circLARP4.

Human

HL

cellular senescence

30,520,539

Gene

0

1

0

1

0

1

0

MIR181A1

406995

ncRNA

ULM,MM

--

Uterine leiomyomas

Accelerate

SA--gal activity assay

ULM and MM cells overexpressing the miRNAs and cultured as spheroids were fixed and stained for SA-¦Â-galactosidase. Spheroid MM and ULM showed a global senescence pattern and a significant increase in cellular senescence was observed in both ULM and MM spheroids with overexpressing miR-29b, miR-181a, miR-182, and miR-200c compared to vector controls.miR-181a and miR-182 overexpression resulted in an intense and diffusive pattern of ¦Â-galactosidase staining in ULM.

Akt3//CCND2

Downregulation//Downregulation

Luciferase reporter assay//Western blot

MiR-181a overexpression can reduce the luciferase activity when transfection of luciferase with AKT3 3 -UTR in myometrial cells Transienttransfection of miR-181a in MM and ULM cells consistently reduced AKT3 expression.MiR-182 overexpression consistently represses CCND2 expression in all MM and LM cells tested.

--

Human

HL

cellular senescence

30,097,674

Gene

0

1

0

MIR182

406958

ncRNA

ULM,MM

--

Uterine leiomyomas

Accelerate

SA--Gal activity assay

ULM and MM cells overexpressing the miRNAs and cultured as spheroids were fixed and stained for SA-¦Â-galactosidase. Spheroid MM and ULM showed a global senescence pattern and a significant increase in cellular senescence was observed in both ULM and MM spheroids with overexpressing miR-29b, miR-181a, miR-182, and miR-200c compared to vector controls.miR-181a and miR-182 overexpression resulted in an intense and diffusive pattern of ¦Â-galactosidase staining in ULM.

Akt3//CCND2

Downregulation//Downregulation

Luciferase reporter assay//Western blot

MiR-181a overexpression can reduce the luciferase activity when transfection of luciferase with AKT3 3 -UTR in myometrial cells Transienttransfection of miR-181a in MM and ULM cells consistently reduced AKT3 expression.MiR-182 overexpression consistently represses CCND2 expression in all MM and LM cells tested.

--

Human

HL

cellular senescence

30,097,674

Gene

0

1

0

MIR26B

407017

ncRNA

Fibroblast

--

Aging

Accelerate

RT-PCR

RT-PCR analysis confirmed a gradual increase in the expression levels of each SA-miRNA with increase in population doublings in fibroblasts.In addition,transient overexpression of each individual SA-miRNAs in fibroblasts also reduced cell proliferation and increased p16 levels.

p16

--

Knockdown

Stable knockdown of p16(HMEC.p16shRNA) generated a relative increase in PcG member expression, particularly in the case ofCBX7. Concordantly, SA-miRNA levels were reduced.We examined the impact of the transient loss of p16 on PcG and SA-miRNA expression.Transient transfection of presenescent HMECs with a potent siRNA targeting p16 increased the expression of each PcG member and repressed SA-miRNA levels in HMEC to a great degree than the changes observed.

--

Human

HL

cellular senescence

24,217,920

Gene

0

1

0

MIR181A1

406995

ncRNA

Fibroblast

--

Aging

Accelerate

RT-PCR

RT-PCR analysis confirmed a gradual increase in the expression levels of each SA-miRNA with increase in population doublings in fibroblasts.In addition,transient overexpression of each individual SA-miRNAs in fibroblasts also reduced cell proliferation and increased p16 levels.

p16

--

Knockdown

Stable knockdown of p16(HMEC.p16shRNA) generated a relative increase in PcG member expression, particularly in the case ofCBX7. Concordantly, SA-miRNA levels were reduced.We examined the impact of the transient loss of p16 on PcG and SA-miRNA expression.Transient transfection of presenescent HMECs with a potent siRNA targeting p16 increased the expression of each PcG member and repressed SA-miRNA levels in HMEC to a great degree than the changes observed.

--

Human

HL

cellular senescence

24,217,920

Gene

0

1

0

MIR210

406992

ncRNA

Fibroblast

--

Aging

Accelerate

RT-PCR

RT-PCR analysis confirmed a gradual increase in the expression levels of each SA-miRNA with increase in population doublings in fibroblasts.In addition,transient overexpression of each individual SA-miRNAs in fibroblasts also reduced cell proliferation and increased p16 levels.

p16

--

Knockdown

Stable knockdown of p16(HMEC.p16shRNA) generated a relative increase in PcG member expression, particularly in the case ofCBX7. Concordantly, SA-miRNA levels were reduced.We examined the impact of the transient loss of p16 on PcG and SA-miRNA expression.Transient transfection of presenescent HMECs with a potent siRNA targeting p16 increased the expression of each PcG member and repressed SA-miRNA levels in HMEC to a great degree than the changes observed.

--

Human

HL

cellular senescence

24,217,920

Gene

0

1

0

MIR424

494336

ncRNA

Fibroblast

--

Aging

Accelerate

RT-PCR

RT-PCR analysis confirmed a gradual increase in the expression levels of each SA-miRNA with increase in population doublings in fibroblasts.In addition,transient overexpression of each individual SA-miRNAs in fibroblasts also reduced cell proliferation and increased p16 levels.

p16

--

Knockdown

Stable knockdown of p16(HMEC.p16shRNA) generated a relative increase in PcG member expression, particularly in the case ofCBX7. Concordantly, SA-miRNA levels were reduced.We examined the impact of the transient loss of p16 on PcG and SA-miRNA expression.Transient transfection of presenescent HMECs with a potent siRNA targeting p16 increased the expression of each PcG member and repressed SA-miRNA levels in HMEC to a great degree than the changes observed.

--

Human

HL

cellular senescence

24,217,920

Gene

0

1

0

MIR194-1

406969

ncRNA

HUVEC

--

Aging

Accelerate

BrdU assay//DAPI staining//SA--gal activity assay//Western blot//qRT-PCR

The increase of miR-194 expression in replicative senescence was validated in human umbilical vein epithelial cells (HUVECs) as well. In order to further confirm the function of miR-194 on cellular senescence, we transfected MEFs with miR-194 inhibitor, Antagomir 194 or Antagomir NC. The results unveiled that miR-194 inhibition by Antagomir 194 transfection conferred attenuated senescence phenotype and enhanced proliferation of MEFs. Consistently, the data of SA-¦Â-gal staining and BrdU assay showed that increased expression of miR-194 conferred NIH/3T3 cells with enhanced H2O2-induced senescence and diminished cell proliferation. In contrast, inhibition of miR-194 protected NIH/3T3 cells against H2O2 induced senescence.

DNMT3A

Downregulation

Dual-Luciferase reporter assay//Western blot

We further constructed a luciferase reporter vector carrying mutant fragment of DNMT3A-3¡¯UTR-2. The results showed that mutation of the miR-194 binding site in the DNMT3A 3¡¯UTR (mut-DNMT3A-2) significantly reversed the Agomir 194-dependent reduction of luciferase reporter activity.Subsequently, we examined the DNMT3A mRNA and protein expression levels in MEFs after transfection with Agomir NC or Agomir 194. The data showed that Agomir 194 transfection significantly suppressed the expression of endogenous DNMT3A at both mRNA and protein levels.

--

Human

L

cellular senescence

27,981,676

Gene

0

1

0

1

0

1

0

1

0

1

0

MIR22

407004

ncRNA

MRC-5,Fibroblast

--

Breast cancer

Accelerate

BrdU assay//Cell proliferation assay//DAPI staining//Flow cytometry//Western blot//qRT-PCR

Quantitative (q) RT-PCR analysis confirmed that miR-22 expression was increased in senescent TIG-3 and other fibroblasts and even up-regulated by more than fivefold in senescent MRC-5 cells.miR-22 also increased SA-¦Â-gal activity miR-22 overexpression induced cell cycle arrest at G1 phase, accompanied by the decrease in percentages of S phase. The inhibitory effect of miR-22 on cell cycle progression has also been recently reported in other cancer cells.

SIRT1//SP1//CDK6

--// --// --

MiRanda,TargetScan,and PicTar//Luciferase reporter assay//Western blot//SA-¦Â-gal activity assay

The mRNAs of SIRT1 and Sp1 contain putative binding sites for miR-22 in their 3-UTRs, and each site is broadly conservative among mammals. CDK6 mRNA contains three miR-22 binding sites in the 3'-UTR, whereas it is different in conservation for each site.In SiHa and MDA-D3 cells, miR-22 significantly reduced the luciferase activities of the full-length 3'-UTR or WT SIRT1, SP1, and CDK6 site 1 and site 3) reporters, compared with the negative cont miR. Western blot analysis showed that overexpression of miR-22 markedly down-regulated SIRT1, SP1, and CDK6 in SiHa and MDA-D3 cells.We forced SiHa or MDA-D3 cells to express SIRT1, Sp1, or CDK6 using plasmid constructs lacking 3ªÚ-UTRs of these genes. Indeed, miR-22¨Cinduced cell growth repression and SA-¦Â-gal activity were partially rescued by the introduction of SIRT1, CDK6, or Sp1 in either SiHa cells or MDA-D3 cells (not depicted), although it seemed weak for the effect of Sp1 overexpression on cell growth, which might be because of the indirect role of Sp1 in the pRb pathway of senescence.

--

Human

HL

cellular senescence

21,502,362

Gene

0

1

0

1

0

1

0

1

0

1

0

1

0

MIR24-1

407012

ncRNA

HDF

--

Aging

Accelerate

SA--gal activity assay

Notably a significantly higher percentage of senescent cells was observed in mir-24 transfected cells.

TOP1

Downregulation

Western blot

This initial finding could be experimentally corroborated by showing that mir-24 overexpression results in TOP1 protein downregulation.

--

Human

L

cellular senescence

26,748,253

Gene

0

1

0

TERC

7012

ncRNA

2BS

--

Aging

Accelerate

Immunostaining//SA--gal activity assay

hTERC-53 overexpressing cells showed a significantly faster senescence rate. Full length hTERC overexpressing cells also showed a similar phenotype, even though to a lesser extent, possibly the result of hTERC-53 accumulation due to overexpression of the full length RNA. Expression of hTERC-53r led to minor downregulation of the expression levels of the senescence markers such as p-AMPK and p16, and removal of the inhibitory modification of MnSOD.

--

Human

HL

cellular senescence

30,788,732

Gene

0

1

0

1

0

MIR34A

407040

ncRNA

HepG2,SMMC-7721,HHCC,SK-Hep-1

--

Hepatocellular carcinoma

Accelerate

Cell morphological analysis//SA--gal activity assay

We observed that introduction of miR-34a into liver cancer cells caused senescence-like phenotypes, with positive staining for senescence-associated ¦Â-galactosidase (SA ¦Â-gal) and enlarged cellular size.

c-Myc//FoxM1//p53

Upregulation//Upregulation//--

qRT-PCR//Western blot//Luciferase reporter assay

The data showed that among the hTERT activators examined, FoxM1 and c-Myc were the most down-regulated genes by miR-34a overexpression.By target prediction, it turned out that FoxM1 and c-Myc have predicted binding sites for miR-34a in their 3 UTRs. With the transfection of miR-34a duplex (miR-34a) in cancer cell lines, we found that miR-34a reduced the protein levels of c-Myc and FoxM1 significantly.Our results showed that miR-34a inhibited luciferase activity significantly, whereas no effect was observed when their respective target sites were mutated, suggesting that miR-34a directly targets c-Myc and FoxM1 via binding to the 3UTRs in liver cancer cells.The miR-34a expression positively correlated with the p53 level (P < 0.05), which was consistent with the NCI-60 data.Our expectation, the p53 protein expression significantly increased after treatment with H2 O2 or cisplatin in the wildtype p53 cells and the levels of miR-34a also increased accordingly.Further, when endogenous p53 expression was knocked down by a p53 siRNA in the four p53 wild-type liver cancer cells, miR-34a expression was also attenuated.

FOXM1-c-Myc

--

qRT-PCR//Western blot

The effect of c-Myc overexpression rescued the FoxM1-mediated inhibition of hTERT, as revealed by qRT-PCR and western blot, suggesting c-Myc as one of the most important downstream factors of FoxM1. As altered expression of miR-34a would contribute to the impaired telomerase activity, we discovered similar effects of miR-34a mimics and FoxM1 siRNA on the hTERT expression, with possible synergistic effect when transfected together, as revealed by qRT-PCR and western blot. With no surprise, similar effects were also observed for miR34a mimics and c-Myc siRNA.

Human

L

cellular senescence

25,686,834

Gene

0

1

0

1

0

MIR433

574034

ncRNA

A2780

--

Ovarian cancer

Accelerate

SA--gal activity assay//Western blot

Firstly,to investigate if miR-433 overexpression could induce senescence the protein levels of p16, phosphorylated Rb (p-Rb), and p21 were anaylzed in the miR-433-stable A2780 cells. This analysis showed that p-Rb was decreased in A2780 cells stably transfected with miR-433. Notably, there was no reciprocal upregulation of p16 and p21 in these cells . The senescence-associated ¦Â-galactosidase activity in these cells was determined by Western blot and ¦Â-galactosidase staining analyses and revealed a significant upregulation of senescence-associated ¦Â-galactosidase activity in miR-433-stable cells compared to controls(P < 0.001).

CDK6

Downregulation

Western blot

In our earlier bioinformatics analysis of potential miR-433 targets, CDK6 was predicted by five of the seven databases as a candidate miR-433 target gene. Therefore, we set out to establish if miR-433 could regulate the expression of CDK6. By analyzing protein expression in both the miR-433 stable A2780 cells and the clonal derivative of this cell line, we observed a decrease in CDK6 expression. Additionally, transient overexpression of miR-433 in HeLa cells also demonstrated downregulation of CDK6. Moreover, the transient transfection of PEO1 cells with anti-miR-433 to inhibit miR-433, resulted in a demonstrable upregulation of CDK6.

--

Human

L

cellular senescence

25,684,390

Gene

0

1

0

1

0

MIR186

406962

ncRNA

HCT116

--

Colorectal cancer

Accelerate

SA--gal activity assay

To examine the role of miR-186, miR-216b, miR-337-3p, and miR-76 in cellular senescence, we knocked down CKIIa in HCT116 cells by co-treatment with the same miRNA mimics.To assess the effect of miRNA knockdown on senescence, after transfection for 2 days the transfectants were then stained for SA-¦Â-gal activity. HCT116 cells transfected with the four mimics exhibited a higher rate of SA-¦Â-gal staining compared with control cells .

CKII//p53//p21/WAF1

Downregulation//Upregulation//Upregulation

CKII activity assay//Western blot

When CKII activity was assessed using [c32P]ATP and CKII peptide substrate, extract from the cells transfected with the four mimics contained lower than 40% CKII activity compared to control extract.Immunoblot data showed that the expression levels of p53 and p21Cip1/WAF1 were upregulated in HCT116 cells treated with the four miRNA mimics in comparison with control cells.

--

Human

HL

cellular senescence

23,137,536

Gene

0

1

0

MIR216B

100126319

ncRNA

HCT116

--

Colorectal cancer

Accelerate

SA--gal activity assay

To examine the role of miR-186, miR-216b, miR-337-3p, and miR-76 in cellular senescence, we knocked down CKIIa in HCT116 cells by co-treatment with the same miRNA mimics.To assess the effect of miRNA knockdown on senescence, after transfection for 2 days the transfectants were then stained for SA-¦Â-gal activity. HCT116 cells transfected with the four mimics exhibited a higher rate of SA-¦Â-gal staining compared with control cells.

CKII//p53//p21/WAF1

Downregulation//Upregulation//Upregulation

CKII activity assay//Western blot

When CKII activity was assessed using [c32P]ATP and CKII peptide substrate, extract from the cells transfected with the four mimics contained lower than 40% CKII activity compared to control extract.Immunoblot data showed that the expression levels of p53 and p21Cip1/WAF1 were upregulated in HCT116 cells treated with the four miRNA mimics in comparison with control cells.

--

Human

HL

cellular senescence

23,137,536

Gene

0

1

0

MIR337

442905

ncRNA

HCT116

--

Colorectal cancer

Accelerate

Knockdown//SA--gal activity assay

To examine the role of miR-186, miR-216b, miR-337-3p, and miR-76 in cellular senescence, we knocked down CKIIa in HCT116 cells by co-treatment with the same miRNA mimics.To assess the effect of miRNA knockdown on senescence, after transfection for 2 days the transfectants were then stained for SA-¦Â-gal activity. HCT116 cells transfected with the four mimics exhibited a higher rate of SA-¦Â-gal staining compared with control cells .

CKII//p53//p21/WAF1

Downregulation//Upregulation//Upregulation

CKII activity assay//Western blot

When CKII activity was assessed using [c32P]ATP and CKII peptide substrate, extract from the cells transfected with the four mimics contained lower than 40% CKII activity compared to control extract.Immunoblot data showed that the expression levels of p53 and p21Cip1/WAF1 were upregulated in HCT116 cells treated with the four miRNA mimics in comparison with control cells .

--

Human

HL

cellular senescence

23,137,536

Gene

0

1

0

1

0

MIR760

100126348

ncRNA

HCT116

--

Colorectal cancer

Accelerate

SA--gal activity assay

To examine the role of miR-186, miR-216b, miR-337-3p, and miR-76 in cellular senescence, we knocked down CKIIa in HCT116 cells by co-treatment with the same miRNA mimics.To assess the effect of miRNA knockdown on senescence, after transfection for 2 days the transfectants were then stained for SA-¦Â-gal activity. HCT116 cells transfected with the four mimics exhibited a higher rate of SA-¦Â-gal staining compared with control cells.

CKII//p53//p21/WAF1

Downregulation//Upregulation//Upregulation

CKII activity assay//Western blot

When CKII activity was assessed using [c32P]ATP and CKII peptide substrate, extract from the cells transfected with the four mimics contained lower than 40% CKII activity compared to control extract.Immunoblot data showed that the expression levels of p53 and p21Cip1/WAF1 were upregulated in HCT116 cells treated with the four miRNA mimics in comparison with control cells.

--

Human

HL

cellular senescence

23,137,536

Gene

0

1

0

HOTAIR

100124700

ncRNA

A2780_CR5

--

Ovarian cancer

Accelerate

Flow cytometry//Knockdown//PI staining//SA--gal activity assay

In addition, IL-6 secretion, an established marker for cell senescence was increased by CDDP in A2780 p overexpressing HOTAIR compared with vector-transfected cells .DsiRNA knockdown of HOTAIR in A2780_CR5 cells reduced the number of senescent cells.We observe a decrease in S1 and an increase in G2 phase 48 h post CDDP treatment in HOTAIR expressing cells and a reversal of this effect in A2780_CR5 cells, suggesting that a subpopulation of cells undergo HOTAIR-dependent cell senescence.

--

NF-¦ÊB//CHK1-p53-p21

--//--

Western blot//SA-¦Â-gal activity assay//MTT assay

In HOTAIR overexpressing A2780p cells, SA-¦Â-Gal-positive cell numbers were increased by high vs low evels of CDDP , and NF-¦ÊB inhibitor Bay-11 reduced the number of senescent cells,ctopic expression of HOTAIR in NF-¦ÊB knockdown cells rescued (Po0.05) proliferation and increased clonogenic survival.In HOTAIR overexpressing vs vector control cells, decreased p-p53 levels were observed and the level of p-Chk1 was essentially unchanged , suggesting ATR dependent activation of p53 by HOTAIR. Activation of p53 and p21 by HOTAIR was observed only during high CDDP treatment, indicated by p53 phosphorylation and p21 expression .

Human

HL

cellular senescence

27,041,570

Gene

0

1

0

1

0

1

0

1

0

MIR145

406937

ncRNA

T24

--

Urothelial carcinoma

Accelerate

SA--gal activity assay//TUNEL assay

miR-145 overexpression significantly suppressed cell proliferation. We found senescence to be induced in overexpressing T24 cells, although the number of apoptotic cells was not significantly increased .

syndecan-1

Downregulation

qRT-PCR

Overexpression of miR-145 significantly suppressed syndecan-1 mRNA.

--

Human

L

cellular senescence

26,514,209

Gene

0

1

0

1

0

MIR23A

407010

ncRNA

K562

--

Chronic myeloid leukemia

Accelerate

SA--gal activity assay

Strongly positive ¦Â-gal staining was observed in K562 cells transfected with miR-23a precursors compared with controls and the difference was statistically significant.

--

BCR-ABL-PI3K-Akt-MMP9

--

qRT-PCR//Western blot//Luciferase reporter assay

Transfection with miR-23a precursor decreased the BCR/ABL 3¡ä-UTR reporter activity in K562 cells, which strongly indicated that BCR/ABL was a target for miR-23a.Finally, we assessed the effect of miR-23a expression on BCR/ABL expression. Transfection of K562 cells with miR-23a precursor resulted in lower expression of BCR/ABL after 48h. Concomitant with decreased BCR/ABL expression, we observed reduction of its downstream targets PI3K, Akt and MMP-9, as we previously reported8.

Human

HL

cellular senescence

25,213,664

Gene

0

1

0

MIR221

407006

ncRNA

Fibroblast

--

Aging

Accelerate

Flow cytometry//qPCR

The impact of miR-221/222 on cell proliferation was investigated by FACS analysis and showed that miR-221/222 promoted the accumulation of HDFs in G1/S cell cycle phase. We then analysed the expression of genes implicated in cell cycle progression in miR-221/222 HDFs. Transient expression of miR-221/222 in both HFL-1 PDL28 and MRC-5 PDL31 HDFs resulted in a reduction in the expression of E2F1 and Cdc6, and in the up-regulation of p21Cip1.

--

Human

HL

cellular senescence

28,658,612

Gene

0

1

0

1

0

MIR222

407007

ncRNA

Fibroblast

--

Aging

Accelerate

Flow cytometry//qPCR

The impact of miR-221/222 on cell proliferation was investigated by FACS analysis and showed that miR-221/222 promoted the accumulation of HDFs in G1/S cell cycle phase. We then analysed the expression of genes implicated in cell cycle progression in miR-221/222 HDFs. Transient expression of miR-221/222 in both HFL-1 PDL28 and MRC-5 PDL31 HDFs resulted in a reduction in the expression of E2F1 and Cdc6, and in the up-regulation of p21Cip1.

--

Human

HL

cellular senescence

28,658,612

Gene

0

1

0

1

0

H19

283120

ncRNA

NPC

--

Disc degenerative disease

Accelerate

Immunofluorescence//SA--gal activity assay

SA-¦Â-Gal positive cells and Collagen I content were increased, while telomerase activity was decreased by H19 overexpression.

miR-22

Downregulation

Luciferase reporter assay//qPCR

In NPCs, H19 and miR-22 negatively regulated each other. The results showed that the luciferase activity of wild-type vectors, wt-19 and wtLEF1 3 UTR could be suppressed by miR-22 mimics while amplified by miR-22 inhibitor; after mutating the predicted miR-22 binding site, the changes of luciferase activity were totally eliminated.

Wnt-¦Â-catenin

--

Spearman's rank correlation analysis

Furthermore, H19 expression was positively correlated Wnt3 and ¦Â-catenin expression in tissue specimens, respectively.The data indicate that H19 modulates Wnt signaling pathway through miR-22; miR-22 could partially reverse the effect of H19 on Wnt signaling pathway.

Human

L

cellular senescence

29,520,849

Gene

0

1

0

1

0

MIR107

406901

ncRNA

HUVEC

--

Aging

Accelerate

Cell cycle analysis

Cell cycle analysis showed a significant increase in the percentage of cell population arrested at G0/G1 phase in the mimic miR-107 transfected cells compared to the mimic negative control cells.Moreover, the percentage of cell population in S-phase was also significantly lower in the mimic miR-107 transfected cells compared to the mimic negative control cells.

p16NK4A//PTEN

Upregulation//Downregulation

Western blot//RNA Pull-down assay//qRT-PCR

Over-expression of miR-107 also resulted in significant augmentation of the expression of P16INK4A .For further verification, miRNA-mRNA complex pull-down assay was performed.Accordingly, qRT-PCR analysis demonstrated a significant enrichment of PTEN mRNA in the pull-down miRNA-mRNA complex . In addition, PTEN protein expression was down-regulated following the over-expression of miR-107 similar to that observed in the 1 nM rapamycin-treated pre-senescent HUVECs.

--

Human

HL

cellular senescence

29,857,052

Gene

0

1

0

RP11-670E13.6

--

ncRNA

HDF

--

Aging

Prevent

Cell cycle analysis//Flow cytometry//Knockdown//SA--gal activity assay

RP11-670E13.6 expression was knocked down using an siRNA, which significantly decreased the proliferation of HDFs.Our analysis revealed that RP11-670E13.6-depleted HDFs showed elevated SA¦Â-gal activity, a distinguishing marker of senescent cells, but no significant effects on apoptosis were observed.RP11-670E13.6 knockdown significantly inhibited cell cycle progression, as evidenced by a significant increase in the G0/G1 fraction and a significant decrease in G2/M cells.

--

p16-pRb

--

Western blot//RT-PCR

RP11-670E13.6 knockdown increased expression of p16£¬RP11-670E13.6 knockdown promoted cell cycle arrest and senescence, perhaps through the p16-pRB pathway, independently of the p53-p21 pathway¡£Knockdown of RP11-670E13.6 had a significant effect on cell senescence-associated mRNA expression. Knockdown of RP11-670E13.6 had a significant effect on cell senescence associated protein expression.

Human

HL

cellular senescence

29,143,326

Gene

0

1

0

1

0

1

0

1

0

MIR138-1

406929

ncRNA

SN-12

--

Renal carcinoma

Accelerate

SA--gal activity assay

Ninety-six hours after introducing the miR-138 mimic, the results revealed that overexpression of miR-138 significantly increased the number of SA-¦Â-gal-positive cells compared with those of the scrambled miRNA mimic.

E2H2

Downregulation

Luciferase reporter assay//SA-¦Â-gal activity assay//Knockdown

The transfection of this reporter construct in the presence of miR-138 mimic induced nearly a 50% downregulation of relative luciferase activity; this effect was abolished with the mutation of miR-138 at the putative target site.Knockdown of EZH2 induced senescence in SN-12 cells.

--

Human

HL

cellular senescence

24,406,044

Gene

0

1

0

MIR22

407004

ncRNA

EPC

--

Aging

Accelerate

MTT assay//SA--gal activity assay

Using ¦Â-galactosidase expression as an indicator of cell senescence, we found that overexpression of miR-22 in Y-EPCs increased senescence.Overexpression of miR-22 in Y-EPCs inhibited proliferation.

Akt3

Downregulation

Luciferase reporter assay//Western blot//RT-PCR

miR-22 overexpression decreased the relative luciferase activity of the 3¡äUTR reporter, whereas it had no effect on the mutant 3¡äUTR reporter, indicating that the intact seed sequence is required for miR-22 functionality.miR-22 overexpression in Y-EPCs significantly downregulated AKT3 mRNA and protein expression.

--

Human

L

cellular senescence

25,323,119

Gene

0

1

0

1

0

MIR181A1

406995

ncRNA

CD4+T

--

HCV infection

Accelerate

EdU assay//SA--gal activity assay

MiR-181a reconstitution significantly reduced the telomere length, increased SA-¦Â-gal+ T cell frequency, and decreased EdU incorporation, in CD4+ T cells from patients with HCV.

SIRT1

Downregulation

Flow cytometry

Transfection of miR-181a precursor, but not the negative control, led to a significant down-regulation of Sirt1 expression in CD4+ T cells.

--

Human

L

cellular senescence

27,354,409

Gene

0

1

0

1

0

MIR503

574506

ncRNA

HPNE

--

Pancreatic cancer

Prevent

Colony formation assay//Flow cytometry//SA--gal activity assay

miR-503 mimic significantly promoted anchoragedependent colony formation, accelerated cell cycle progression, and decreased KRASG12D-induced senescence in HPNE/KRASG12D/shNC cells.miR-503 inhibitor significantly inhibited anchorage-dependent colony formation, retarded cell cycle progression, and increased cell senescence in HPNE/KRASG12D/shARID1A.

p16

Downregulation

Western blot

The expression of p16 was obviously decreased in HPNE/KRASG12D/ shNC cells upon the transfection of miR-503 mimic, whereas miR503 inhibitor led to increased p16 expression in HPNE/KRASG12D/ shARID1A cells, as shown by western blot assay.

--

Human

L

cellular senescence

28,942,143

Gene

0

1

0

1

0

1

0

MIR146A

406938

ncRNA

HUVEC

--

Aging

Prevent

SA--gal activity assay

In P5 HUVEC, we found that miR-146a overexpression decreased the percentage of SA-¦Â-gal-positive cells compared with control at 3 days after transfection.

--

Human

L

cellular senescence

21,511,256

Gene

0

1

0

MIR10A

406902

ncRNA

MSC

--

Aging

Prevent

Knockdown//SA--gal activity assay

Down regulation of miR-10a expression increased the percentage of SA-¦Â-gal positive cells among the LV-anti-10a-infected groups compared to the LV-controlinfected groups, indicating that repression of miR-10a increased hMSC senescence in both young and old hMSCs.

KLF4

Downregulation

Luciferase reporter assay//Western blot//qRT-PCR

Luciferase activity was significantly repressed by the miR-10a mimic, proving the direct binding of the miR-10a to the 30 -UTR of KLF4. A similar effect was also observed in the hMSCs.To determine if miR-10a would affect endogenous KLF4 expression, we compared KLF4 expression after infecting the hMSCs with LV-miR-10a and LVanti-10a. Up regulation of miR-10a repressed both the endogenous mRNA and protein expression of KLF4.

--

Human

HL

cellular senescence

23,696,417

Gene

0

1

0

1

0

MIR34A

407040

ncRNA

HASMC

--

Cardiovascular disease

Accelerate

Flow cytometry//SA--gal activity assay//Western blot

In comparison with the control (miRNA mimic control [SCR]), miR-34a mimic reduced cell number already by 48 hours after transfection;the main and statistically significant differences were observed at 72- hours post-transfection. Further, we evaluated cell cycle distribution by fluorescence-activated cell sorting analysis after propidium iodide (PI) staining. miR-34a ectopic expression significantly increased the percentage of HASMCs in G0¨CG1 phase at 24-hours post-transfection when compared with negative control.Accordingly, p21 protein levels were higher in miR-34a-overexpressing cells.We found that miR-34a enhanced the percentage of SA-¦Â-gal-positive cells at 72 hours after transfection, while HASMCs transfected with the negative control did not show any difference when compared with untransfected cells .

SIRT1

Downregulation

Western blot

We then performed a rescue experiment by transfecting replicative cells with miR-34a mimic or mimic negative control along with either a 3?-untraslated region-deleted-SIRT1-expression vector (devoid of the sequences matching miR-34a seed sequence) or the corresponding empty vector. The WB analysis confirmed that endogenous SIRT1 protein levels were severely lowered upon miR-34a overexpression, while exogenous SIRT1 protein levels were unaffected.

--

Human

L

cellular senescence

25,352,462

Gene

0

1

0

1

0

1

0

MIR152

406943

ncRNA

HDPSC

--

Aging

Accelerate

MTT assay//SA--gal activity assay

The MTT assay showed that miR152 upregulation significantly decreased the viability of HDPSCs.Furthermore, we found that miR-152 overexpression significantly increased the number of SA-¦Â-gal-positive cells concomitant with a significant increase in p21 and p16 expression levels compared with vector control cells.

SIRT7

Downregulation

Knockdown//Western blot//qRT-PCR//Luciferase reporter assay

Transduction of late-passaged HDPSCs with a lentiviral vector encoding anti-miR-152 effectively downregulated miR-152 and upregulated SIRT7.Luciferase activity assays showed that miR-152 significantly decreased luciferase activity in cells transfected with the wild-type but not the mutant SIRT7 3'-UTR-binding site, confirming that SIRT7 is a direct target of miR-152. Real time RT-PCR and western blot analysis showed that SIRT7 was significantly downregulated in HDPSCs at PD54 compared with its expression at PD16, and significantly downregulated by ectopic expression of miR-152, further supporting SIRT7 as a direct target of miR-152.

--

Human

L

cellular senescence

26,991,832

Gene

0

1

0

1

0

MIR21

406991

ncRNA

U87

--

Glioma

Prevent

Knockdown//MTT assay//SA--gal activity assay

Compared with the blank and NC groups, the miR-21 mimics and siRNA-SPRY1 groups revealed a decreased density of U87 cells and enlarged cell size, in addition to reduced senescence indexes. In the miR-21 inhibitors group, the senescence index of U87 cells was increased; and no significant difference in senescence index was identified in the miR-21 inhibitors + siRNA-SPRY1 group.MTT results indicated that, compared with the blank and NC groups, cell proliferation reduced in the miR-21 inhibitors group and increased in the miR-21 mimics and siRNA-SPRY1 group; there were no significant differences in cell proliferation in the miR-21 inhibitors + siRNA-SPRY1 group.

SPRY1

Downregulation

Dual-Luciferase reporter assay//qRT-PCR

The results of the dual-luciferase reporter assay system indicated that there was a significant reduction in the luciferase signal in SPRY1-wt cotransfection group, compared with other miR-21 transfection groups. And, there was no difference in the luciferase signal of SPRY1-mut among miR-21 transfection groups.The results of RT-qPCR showed that, compared with the control group, mRNA levels of miR-21, PI3K, and AKT increased in the glioma group, but mRNA levels of SPRY1 and PTEN decreased.

PTEN-PI3K-AKT

--

Western blot//RT-PCR

In the miR-21 mimics and siRNA-SPRY1 groups, protein levels of PI3K, AKT, p-AKT, P53, and p-GSK3 increased, and those of SPRYI1, PTEN, Caspase-3, and Caspase-9 decreased;and the miR-21 inhibitors group exhibited opposite trends; in the miR-21 mimics group, miR-21 expression and mRNA expressions of PI3K and AKT increased while mRNA levels of SPRY1 and PTEN decreased .

Human

L

cellular senescence

29,316,313

Gene

0

1

0

1

0

1

0

MIR449A

554213

ncRNA

95D,A549

--

Lung cancer

Accelerate

EdU assay/SA--gal activity assay//Flow cytometry

MiR-449a introduction caused a remarkable inhibition of cell growth in both A549 and 95D cells relative to NC. The EdU incorporation assay also revealed that the growth of A549 and 95D cells was significantly inhibited 231 by miR-449a relative to NC . The EdU cell proliferation as- 232 say determined that miR-449a suppressed the entry of A549 and 233 95D cells into the S phase.The miR-449a mimics caused significant G0/G1 arrest in A549 and 95D cells. We also observed that miR-449a in A549 and 95D cells caused senescent phenotypes with positive staining for senescence-associated ¦Â-galactosidase (SA-¦Â-gal) and led to drastic changes in cell morphology, including increased size and a broad, flattened shape 72 h following transfection.

E2F3

Downregulation

Dual-Luciferase reporter assay//qRT-PCR//Western blot

The luciferase reporter assay indicated that the luciferase activity of the re porter containing the E2F3 gene¡¯s wide-type 3' -UTR decreased (52%) following treatment with miR-449a mimics. By contrast, the inhibitory effect of the miR-449a mimics was abolished in the mutated construct. In addition, qRT-PCR and western blot analysis revealed that the expression of E2F3 mRNA and protein was inhibited by treatment with miR-449a mimics in A549 and 95D cells.

--

Human

L

cellular senescence

24,211,326

Gene

0

1

0

1

0

MIR340

442908

ncRNA

GIC

--

Glioma

Accelerate

Flow cytometry//SA--gal activity assay//Western blot

We observed that miR-340-overexpressing cells became flatter and larger than control cells.Immunohistochemical analysis and Western blot assays revealed that miR-340 overexpressing hGICs partially decreased Nestin expression and lost the expression of Sox2, but remained positive for GFAP, an astrocyte marker. miR-340-overexpressing hGICs ceased proliferating during the first 3 days of culture. The BrdU-incorporation and cell cycle analyses revealed that miR-340 overexpression significantly arrested the cell cycle at the G1/S transition, as indicated by a marked accumulation of cells in the G1 peak and by a reduction of cells in the S phase. We further demonstrated that miR-340 overexpression activated the expression of SA-¦Â-gal, a marker of cellular senescence, in hGICs.

PLAT

Downregulation

Luciferase reporter assay//Western blot//Immunohistology

Immunohistochemical analysis and Western blot assays confirmed that miR-340 overexpression decreased PLAT expression in hGICs. Using a reporter vector encoding the firefly luciferase gene with the wild-type plat 3¡¯UTR, we demonstrated that miR-340 overexpression inhibited luciferase activity in hGICs, whereas a deletion in the predicted binding site of miR-340 in the 3¡¯UTR of the plat gene abrogated the aforementioned inhibitory effect of miR-340. Taken together, these data strongly indicate that PLAT is a novel direct target of miR-340 in hGICs.

--

Human

L

cellular senescence

25,627,976

Gene

0

1

0

1

0

1

0

MIR34A

407040

ncRNA

UM-SCC-23,Fadu

--

Squamous cell carcinoma

Accelerate

Flow cytometry//SA--gal activity assay

Compared with the wide type and control cells, the miR-34a over-expression cells exhibited an about 1.6 to 2.0 times increase in the percentage of cell at G0/G1 phase, along with a?decreased cell percentage at the S?phase.The result showed that the mean percentage of SA-¦Â-gal-positive cells increased by 4.41 times (UM-SCC-23-miR-34a) and 8.18 times (Fadu-miR-34a) in miR-34a over-expression cell lines than that in control cell lines.

MAP2K1//AXL//FUT1//AREG

--//--//--//--

Luciferase reporter assay//qRT-PCR

The result indicated that the mRNA levels of 8 genes were reduced about 1.4-fold to 4.7-fold in cells with ectopic miR-34a expression, compared with control.The luciferase reporter activity of pSiCheck?-2-AXL-3¡¯UTR, pSiCheck?-2-FUT1-3¡¯UTR and pSiCheck?-2-MAP2K1-3¡¯UTR was markedly reduced by forced expression of miR-34a compared with the control(2.43- fold decrease, 2.08-fold decrease and 4.78-fold decrease,respectively), whereas no reporter activity were found when their target sites were mutated.

--

Human

HL

cellular senescence

28,485,160

Gene

0

1

0

1

0

MIR125B1

406911

ncRNA

KYSE150,KYSE510

--

Esophageal cancer

Accelerate

CCK-8 assay//SA--gal activity assay//Transwell assay

Overexpression of miR-125b-5p significantly inhibited cell proliferation of KYSE150 and KYSE510 cell lines.Using Senescence ¦Â-Galactosidase Staining Assay, we found that overexpression of miR-125b-5p significantly enhanced the senescence of ESCC cells. We also found that miR-125b5p overerxpression significantly inhibited the migration and invasion of ESCC cells.

HMGA2

Downregulation

Western blot//qRT-PCR//Knockdown//Dual-Luciferase reporter assay

HMGA2 was one of the candidate targets, and down-regulated by miR-125b-5p mimics both in mRNA and protein levels. Dual-luciferase reporter assays found that luciferase activity was significantly down-regulated in the miR-125b-5p mimics and pGL3-HMGA2-3¡¯UTR-Wt group compared with NC group.

--

Human

HL

cellular senescence

28,968,424

Gene

0

1

0

1

0

1

0

MIR34A

407040

ncRNA

PPC-1

--

Prostate cancer

Accelerate

Flow cytometry//SA--gal activity assay

MiR-34a overexpression induced significantly increased cell senescence assessed by staining of prostate cancer cells for senescence-associated ¦Â-gal (SA-¦Âgal) activity. It is well documented that G1 cell-cycle arrest generally precedes cell senescence.

--

Human

L

cellular senescence

22,719,071

Gene

0

1

0

1

0

MIR212

406994

ncRNA

HCT116

--

Prostate cancer

Accelerate

SA--gal activity assay

In both SF and CM conditions, we show that transfection with miR-212 resulted in increased cellular senescence as shown by senescence associated ¦Â-galactosidase staining as compared to negative control mimic.

SIRT1

Downregulation

SA-¦Â-gal activity assay

We asked, whether miR-212 could modulate cellular senescence. Using HCT116 cells, we demonstrate that miR-212 inhibit endogenous SIRT1 levels in both serum free (SF) and complete media (CM). Co-transfection with SIRT1 reduced the number of senescent cells in miR-212 transfected cells partially abrogating its senescence inducing affect.

--

Human

L

cellular senescence

26,439,987

Gene

0

1

0

MIR23A

407010

ncRNA

HDF

--

Aging

Accelerate

FACS analysis//Histochemical staining//SA--gal activity assay//qRT¨CPCR

Non-senescent fibroblasts were transfected with the miR-23a-3p mimic and oligonucleotide control. Seven days after the transfection, a threefold increase in SA-¦Â-gal was detected.Inhibition of mir-23a-3p in senescent fibroblasts reduced the senescent phenotype of the cells and rescued the loss of the HA matrix.

HAS2

Downregulation

qRT-PCR//Luciferase reporter assay

Quantitative real-time reverse-transcriptase¨CPCR (qRT-PCR) analysis revealed that miR-23a-3p expression was increased and HAS2 mRNA expression decreased in senescent compared with non-senescent fibroblasts.In skin biopsies from mice aged 18 and 48 weeks, the miR-23a-3p copy number was increased and the HAS2 mRNA expression decreased, as seen in aged and senescent human dermal fibroblasts previously. Transfection of senescent cells, which display high levels of endogenous miR-23a-3p, with the miR-23a-3p inhibitor increased the HAS2 mRNA copy number twofold. Luciferase activity was not decreased when cells were transfected with luciferase constructs containing a mutated version of the HAS2 3¡äUTR binding site and the miR-23a-3p mimic .

--

Human

HL

cellular senescence

25,264,594

Gene

0

1

0

1

0

1

0

1

0

MIR141

406933

ncRNA

MSC

Umbilical cord blood

Aging

Accelerate

MTT assay//SA--gal activity assay

In the miR-141-3p-transfected cells, MTT assays and cell cycle analyses revealed that the growth rate was reduced 48?hours post-transfection. In contrast, the transfection of hMSCs with anti-miR-141-3p yielded an increase in proliferation. The correlation between the expression of ZMPSTE24 and miR-141-3p was confirmed in five other hMSC cell lines .The overexpression of miR-141-3p also increased SA-¦Â-gal activity and ¦ÃH2Ax expression in these cells.

ZMPSTE24

Downregulation

Western blot//Knockdown

We further investigated whether miR-141-3p regulates ZMPSTE24 expression, prelamin A accumulation and nuclear abnormalities. The transfection of hMSCs with miR-141-3p decreased ZMPSTE24 expression, resulting in increased expression of prelamin A, p16INK4A?and ¦ÃH2AX. Conversely, these effects were reversed by miR-141-3p knockdown.

--

Human

L

cellular senescence

24,101,728

Gene

0

1

0

1

0

MIR17

406952

ncRNA

MCF

Heart

Aging

Prevent

Cell apoptosis assay//SA--gal activity assay//qRT-PCR

Transfection of the miR-17-3p inhibitor enhanced MCF senescence and apoptosis in cells treated with H2O2, whereas expression of miR-17-3p repressed senescence and apoptosis.Consistent with these results, we found that the aging hearts expressed significantly lower levels of miR-17-3p compared with the levels in hearts from young animals.

Par4

Downregulation

Luciferase reporter assay//Western blot//SA-¦Â-gal activity assay

Luciferase activity was repressed when the construct (Luc-Par4) was co-transfected with miR-17-3p mimic, and the repression was abolished when the target site was mutated, thus confirming direct targeting of Par4 by miR-17-3p. Protein lysates prepared from miR-17- and vector-transfected cells were subjected to western blotting, which showed that ectopic transfection of miR-17 repressed Par4 expression significantly. Ectopic expression of Par4 promoted senescence when the cells were cultured in serum-free medium or treated with H2O2.

CEBPB-FAK

--

Western blot//IP//RT-PCR//PCR//ChIP

The ChIP assay indicated that Par4 might bind to the CEBPB promoter directly and repress CEBPB transcription, whereas CEBPB could bind to the FAK promoter and enhance FAK transcription. By using western blotting, we confirmed that silencing Par4 promoted the expression of CEBPB and FAK.The Par4-expressing cells showed high levels of Par4 and low levels of CEBPB and FAK, and the regulation appeared to occur at the transcriptional level. In addition, ectopic transfection of CEBPB enhanced FAK expression at the mRNA and protein levels. By ChIP, immunoprecipitated exogenous Par4 was shown to pull down more CEBPB promoter DNA, whereas immunoprecipitated exogenous CEBPB could pull down more FAK promoter DNA.

Human

L

cellular senescence

25,472,717

Gene

0

1

0

1

0

1

0

MIR143

406935

ncRNA

VSMC

--

Aging

Accelerate

SA--gal activity assay

Our results showed that overexpression of miR-143 promoted senescence,and miR-143 inhibitor suppressed senescence induced by H2O2.

--

Human

L

cellular senescence

25,655,189

Gene

0

1

0

MIR449A

554213

ncRNA

B5,PC-3,DU 145,DU-1.1

--

Prostate cancer

Accelerate

Flow cytometry//SA--gal activity assay

We transfected DU-145 sublines with miR-449a and stained for SA-¦Â-gal activity. At 10 and 25 nM concentrations, DU-1.1 and B5 cells stained positive for SA-¦Â-gal, while staining in mock and miR-Con treatments were nearly undetectable . In PC-3 cells, miR-449a caused G0/G1 arrest as indicated by the increase in G0/G1 cell number and corresponding reductions in S and G2/M populations.

Cyclin D1//HDAC1

Downregulation//Downregulation

Western blot//Knockdown

miR-449a significantly reduced Cyclin D1 protein levels in PC-3 cells.Knockdown of HDAC1 by miR-449a or siHDAC1 elevated p27 protein and reduced P-Rb levels.

Rb

Downregulation

Western blot//Knockdown

Knockdown of Cyclin D1 by miR-449a or siCCND1 drastically reduced phophorylated Rb (P-Rb) levels. Knockdown of HDAC1 by miR-449a or siHDAC1 elevated p27 protein and reduced P-Rb levels.

Human

L

cellular senescence

20,948,989

Gene

0

1

0

1

0

MIR21

406991

ncRNA

HUVEC

--

Aging

Accelerate

MTT assay//SA--gal activity assay//qPCR

Stable miR21 over-expression indeed significantly reduced cell proliferation consistent with the transient over-expression results above.Cell growth was further monitored during the entire replicative lifespan, and indeed, miR-21 over-expressing cells underwent replicative senescence earlier than control cells.Furthermore, miR-21 over-expression led to an increase in senescent cells, as the majority of cells stain positive for senescenceassociated ?-galactosidase (SA ?-gal) at PD1pT after selection of stable cells.

CDC25A//NF1B

--//--

Western blot

CDC25A and NF1B are direct targets of miR©\21. The expression of a luciferase reporter gene containing the CDC25A 3¡ä©\UTR (A) or NF1B 3¡ä©\UTR (B), respectively, was suppressed by miR©\21 in HUVEC. The suppression is specific to the miR©\21 seed region within the CDC25A 3¡ä©\UTR or NF1B 3¡ä©\UTR, as mutation of the miR©\21 target sites alleviated repression by miR©\21. Ratios of CDC25A©\ or NF1B©\dependent renilla luciferase signal (rLuc) to a firefly luciferase signal (fLuc) encoded by the same vector are given.

--

Human

HL

cellular senescence

23,496,142

Gene

0

1

0

1

0

1

0

MIAT

440823

ncRNA

MCF-7

--

Breast cancer

Prevent

Flow cytometry//Knockdown//SA--gal activity assay//Western blot

The results showed an increase in the proportion of cells in G1 phase and a decreased in the proportion of cells in G2/M phase in Miat knock down cells.The result revealed that Cyclin D1 expression level was downregulated in cells treated with Miat siRNA in comparison to cells treated with scramble siRNA, which indicated Miat inhibition caused cell cycle arrest. The result revealed that the proportion of senescent cells was dramatically elevated in Miat knockdown cells in comparison to control cells. Consistent with above stain positive for senescence-associated ¦Â-galactosidase activity, the expression of cellular senescent biomarkers, p16Ink4A and Cox2, markedly increased following Miat suppression.

--

Human

L

cellular senescence

29,345,338

Gene

0

1

0

1

0

1

0

1

0

OVAAL

148756

ncRNA

ME4405,HCT116

--

Colorectal cancer

Prevent

Flow cytometry//Knockdown//PI staining//SA--gal activity assay

OVAAL shRNA inhibited cancer cell proliferation, which was substantiated through G0/G1 cell cycle arrest and decreased phosphorylated Rb (p-Rb); depletion of OVAAL also triggered cellular senescence as shown by induction of senescence-associated (SA)¨C?-gal staining .

STK3//p27

Binding//--

Western blot//qPCR//RIP

Further examination of the relationship between OVAAL and p27 expression showed that the increased p27 protein levels observed after silencing of OVAAL did not increase p27 mRNA levels.Consistently, OVAAL was coimmunoprecipitated by STK3 antibodies by RNA immunoprecipitation (RIP) assays in ME4405 and HCT116 cells .

Raf-MEK-ERK

Activation

Western blot

Silencing of OVAAL prominently reduced the activation of RAF/MEK/ERK cascade as shown by decreases in p-MEK, p-ERK, p-MSK1, and p-RSK1, recapitulating the effects observed after knockdown of STK3 in ME4405 and HCT116 cells.

Human

L

cellular senescence

30,478,051

Gene

0

1

0

1

0

1

0

1

0

MIR34A

407040

ncRNA

TIG3 TERT/B-RAF:ER

--

Aging

Accelerate

Cell cycle analysis//Cell morphological analysis

Cells overexpressing miR-34a exhibited a senescence-like morphology.Overexpression of miR-34a reduced cellular proliferation.

B-RAFoncogene//ELK1//Myc

--//Binding//Downregulation

qPCR//Knockdown//Western blot

miR-34a was approximately eightfold upregulated after 3 days of B-RAF activation. ER cells using siRNA and measured miR-34a regulation after B-RAF activation£¬depletion of ELK1 significantly impaired B-RAF-mediated induction of miR-34a(P<0.04, Student's t-test). Strikingly, miR-34a overexpression resulted in marked downregulation of endogenous MYC protein.

--

Human

HL

cellular senescence

19,696,787

Gene

0

1

0

1

0

MIR137

406928

ncRNA

IMR-90

--

Pancreatic cancer

Accelerate

Immunostaining//SA--gal activity assay

A robust senescence response was observed upon transfection of miR-137 compared to miR-67-transfected cells, as assessed using senescence-associated ¦Â-galactosidase activity .an increase in the number of PML nuclear bodies and ¦ÃH2AX foci, two known markers of cells undergoing cellular senescence, also were observed in miR-137-expressing cells.

KDM4A

Downregulation

qRT-PCR//Western blot

The mRNA level of CHD5, a transcriptional target of KDM4A (Mallette and Richard, 2012), was increased following transfection with miR-137, consistent with decreased KDM4A mRNA expression and protein levels.

ARF-p53//p16-pRb

Activation//Activation

SA-¦Â-gal activity assay//qRT-PCR

Expression of human papilloma viral E6 and E7 in miR-137-transfected fibroblasts fully rescued the senescence phenotype, suggesting that miR-137 indeed relies on either the p53 or p16INK4A?pathway to establish senescence.

Human

L

cellular senescence

26,904,954

Gene

0

1

0

1

0

MIR494

574452

ncRNA

HUVEC

--

Aging

Accelerate

Cell cycle analysis//Cell morphological analysis//SA--gal activity assay//Western blot

Gain of miR-494 in early-passage HUVECs increased senescence-associated ¦Â-galactosidase levels (SA-¦Â-gal).Ectopic expression of miR-494 increased G1 arrest, whereas the inhibition of the miR decreased the G2 arrest in response to a 10?Gy dose of radiation .miR-494 impacted other hallmarks of senescence¡ªa decrease in telomerase activity , an increase in the cell cycle regulator p21 as well as a decrease in Rb hyperphosphorylation .phalloidin staining in HUVECs after 48?h of miR-494 treatment also revealed flattened and multinucleated cells.

MRN

Downregulation

qRT-PCR//Western blot//Immunostaining//Luciferase reporter assay

Transfection of miR-494 decreased both MRN RNA and protein levels,miR-494 decreased activity of a luciferase reporter cloned upstream of each 3¡¯-UTR of the MRN complex genes . miR-494-transfected HUVECs also showed a decrease in MRE11a nuclear foci.

--

Human

L

cellular senescence

29,795,397

Gene

0

1

0

1

0

1

0

1

0

MIR221

407006

ncRNA

A549

--

Lung cancer

Accelerate

SA--gal activity assay

A549-miR-221 cells induced cellular senescence following Cisplatin treatment as compared to A549-Cont cells, detected by SA-¦Â-gal activity assay.

--

PTEN-Akt

--

Western blot

When miR-221 was ectopically expressed, PTEN was decreased and Akt activity was increased in H1299 cells. On the other hand, miR-221 antisense oligonucleotides (ASO) were used to suppress miR-221 activity, and upregulation of PTEN and downregulation of Akt activity in A549 cells were accompanied.

Human

L

cellular senescence

29,876,362

Gene

0

1

0

TERC

7012

ncRNA

--

Lung

Inflammation

Prevent

SA--gal activity assay//Western blot

¦Â-Gal staining for cellular senescence was markedly increased in G3 TERC-null lung sections compared with control;We analyzed the levels of heterochromatin protein 1¦Ã(HP1¦Ã), a marker for senescence-associated heterochromatin foci, and found that immunoreActivate HP1¦Ã was also markedly increased, with the level of HP1¦Ã in TERC-/- AECII being more than 4 times that in WT control.

--

Human

L

cellular senescence

26,518,879

Gene

0

1

0

1

0

MIR29C

407026

ncRNA

MSC

--

Aging

Accelerate

CCK-8 assay//EdU assay//SA--gal activity assay//qRT-PCR

The results demonstrated that the over-expression of miR-29c-3p significantly enhanced the SA-¦Â-gal staining compared with that of the negative control (NC) group. EdU was not incorporated into the SA-¦Â-gal positive hMSCs, indicating the senescent hMSCs were indeed not proliferating.the CCK-8 assay was performed ,The results showed that the proliferative capacity of hMSCs was inhibited after the Agomir-29c-3p transfection.The qPCR results indicated that p16, p21, PTGS2, AKAP9, CCND1 and EDN1(senescence markers ) were significantly increased after miR-29c-3p transfection.

CNOT6

--

Western blot//qRT-PCR

We transfected hMSCs with Agomir-29c-3p or mirVana-29c-3p and examined CNOT6 expression using qPCR and WB assays. The results showed that the CNOT6 mRNA level was decreased in the Agomir-29c-3p group, but increased in the mirVana-29c-3p group.

p53-p21//p16-pRb

Upregulation//Upregulation

Western blot//qRT-PCR

Western blot (WB) results also suggested that the expression of p53, p21, p16, PTGS2 and pRB were all increased gradually.

Human

L

cellular senescence

26,792,405

Gene

0

1

0

1

0

1

0

1

0

MIR1236

100302242

ncRNA

A549

--

Lung cancer

Accelerate

Colony formation assay//SA--gal activity assay

Positive ¦Â-galactosidase cells were observed to increase prominently following transfection of the miR-1236.miR-1236 was obviously suppressed the number of colonies.

p21

Upregulation

Flow cytometry//SA-¦Â-gal activity assay//Colony formation assay

Flow cytometry analysis suggested that cotransfection of miR-1236 with siP21 remarkably attenuated Go/G1 cycle arrest.Silencing p21 dramatically inhibited miR-1236 induced cell senescence.The ability of proliferation and colony formation were restored by p21 silencing.

CCND1-CDK4//CCND1-CDK6

Downregulation

qRT-PCR

As is shown, compared to dsControl treatment, positive control (dsP21-322), miR-1236-3p significantly downregulated expression of Cyclin D1 and CDK4/CDK6 mRNA in A549 .

Human

L

cellular senescence

28,631,573

Gene

0

1

0

1

0

MIR370

442915

ncRNA

A549

--

Lung cancer

Accelerate

Colony formation assay//SA--gal activity assay

Positive ¦Â-galactosidase cells were observed to increase prominently following transfection of the miR-370.miR-370 was obviously suppressed the number of colonies.

p21

Upregulation

Flow cytometry//SA-¦Â-gal activity assay//Colony formation assay

Flow cytometry analysis suggested that cotransfection of miR-370 with siP21 remarkably attenuated Go/G1 cycle arrest.Silencing p21 dramatically inhibited miR-370 induced cell senescence.The ability of proliferation and colony formation were restored by p21 silencing.

CCND1-CDK4//CCND1-CDK6

Downregulation

qRT-PCR

As is shown, compared to dsControl treatment, positive control (dsP21-322), miR-370 significantly downregulated expression of Cyclin D1 and CDK4/CDK6 mRNA in A549 .

Human

L

cellular senescence

28,631,573

Gene

0

1

0

1

0

MIR21

406991

ncRNA

HUVEC

--

Aging

Accelerate

SA--gal activity assay

The percentage of ¦Â-galactosidase staining-positive cells in the ox-LDL group was significantly increased compared to that in the control group. The ox-LDL-induced endothelial cell senescence was markedly reduced in the presence of miR-21-5p inhibitor. The negative control of inhibitor did not exert effect on ox-LDL-induced endothelial cell senescence.

Drp1

Downregulation

Western blot//qRT-PCR

Both mRNA and protein expressions of Drp1 were significantly downregulated in the ox-LDL-treated HUVECs, which were reversed in the presence of miR-21-5p inhibitor.

AMPK-p53//AMPK-p16

Activation

Western blot

The phosphorylation level of AMPK(p-AMPK) was obviously elevated in the ox-LDL-treated HUVECs compared to that in the control cells, concomitant with an up-regulation of p53 and p16, indicating an activation of AMPK-p53/p16 pathway.These phenomena were blocked by miR-21-5p inhibitor.

Human

L

cellular senescence

28,347,692

Gene

0

1

0

MIR203A

406986

ncRNA

HUVEC

--

Aging

Accelerate

SA--gal activity assay

The percentage of ¦Â-galactosidase staining-positive cells in the ox-LDL group was significantly increased compared to that in the control group. The ox-LDL-induced endothelial cell senescence was markedly reduced in the presence of miR-203a-3p inhibitor. The negative control of inhibitor did not exert effect on ox-LDL-induced endothelial cell senescence.

Drp1

Downregulation

Western blot//qRT-PCR

Both mRNA and protein expressions of Drp1 were significantly downregulated in the ox-LDL-treated HUVECs, which were reversed in the presence of miR-203a-3p inhibitor.

AMPK-p53//AMPK-p16

Activation

Western blot

The phosphorylation level of AMPK(p-AMPK) was obviously elevated in the ox-LDL-treated HUVECs compared to that in the control cells, concomitant with an up-regulation of p53 and p16, indicating an activation of AMPK-p53/p16 pathway.These phenomena were blocked by miR-203a-3p inhibitor.

Human

L

cellular senescence

28,347,692

Gene

0

1

0

H19

283120

ncRNA

HUVEC

--

Cardiovascular disease

Prevent

Flow cytometry//Knockdown//SA--gal activity assay//Western blot

We first analyzed the role of H19 in proliferation. siRNA-mediated depletion of H19 in HUVECs led to a significant reduction of cells in S- and G2/M-phase, while cells accumulated in G0/G1 phase;silencing of H19 increased the number of acidic ¦Â-galactosidase positive HUVECs and hCoAECs, which is a marker of cellular senescence;Lentivirus-mediated overexpression of H19 tended to reduce p16 and p21 expression;These findings demonstrate that H19 negatively regulates the well-described increase in inflammatory activation in aging.

--

STAT3

Downregulation

qRT-PCR

STAT3 is known to regulate p21 and ICAM-1,the inhibition of STAT3 with Cryptotanshinone (CPT) abolished the H19 depletion-mediated induction of p21. Furthermore, the induction of ICAM-1 and VCAM-1 after H19 depletion was attenuated by STAT3 inhibition as well .

Human

HL

cellular senescence

30,107,531

Gene

0

1

0

1

0

1

0

1

0

MALAT1

378938

ncRNA

Fibroblast

Foreskin

Aging

Accelerate

SA--gal activity assay

The ratio of senescent cells was markedly greater following UVB irradiation (74.4%) compared with cells that had not been exposed to irradiation (15.7%).In addition, the ratio of senescent cells was significantly lower following intervention with MALAT1 siRNA (52.1%) compared with the UVB irradiation group.

--

ERK-MAPK

--

Western blot

MALAT1 siRNA inhibited UVB-induced ERK phosphorylation.

Human

L

cellular senescence

28,487,970

Gene

0

1

0

MIR30C1

407031

ncRNA

BJ

Pancreas

Aging

Prevent

Cell proliferation assay//Growth curve assay//SA--gal activity assay//qRT-PCR

Increased expression of each of the 5 miR-30 family members abrogated induction of proliferative arrest and SA-¦Â-gal by HarasV12 in BJ cells .We detected less SA-¦Â-gal positive epithelial cells in PanIN lesions from the miR-30c-1 transgenic than the control pancreas.

CHD7//TNRC6A//p16//p53

Downregulation//Downregulation//Downregulation//Downregulation

qRT-PCR

All 5 miR-30 members inhibited the expression of endogenous CHD7 and TNRC6A mRNA in BJ cells;Corresponding to reduced senescence in the skin tumors from miR-30c transgenic mice, these tumors contained less cells positive for ¦ÃH2AX or activated p53 (p53-pS15), indicating abrogation of DDR, and less p16INK4A-positive cells, as compared to those from wild-type littermates .

--

Human

L

cellular senescence

29,907,771

Gene

0

1

0

1

0

1

0

1

0

MIR34A

407040

ncRNA

U87,MSC

--

Aging

Accelerate

CCK-8 assay//ELISA//SA--gal activity assay//qRT-PCR

Co-culturing with MSCs overexpressing miR-34a for 24 h impaired U87 glioma cell proliferation. Additionally, it induced expression of senescence-associated genes p53, Cdkn1a and Cdkn2c. As expected,this result was corroborated by an increase in the percentage of cells positively marked with the senescence-associated ¦Â-galactosidase. It was identified that co?culture with hMSCs overexpressing miR-34a decreased the telomere length and impaired the telomerase activity.

SIRT1

Downregulation

Western blot

The results confirmed that the level of SIRT1 decreased in U87 cells co-cultured with hMSCs overexpressing miR-34a, while NC mimic transfection had no observable effect.

--

Human

L

cellular senescence

30,592,284

Gene

0

1

0

1

0

1

0

1

0

MIR34A

407040

ncRNA

ADSC

--

Aging

Accelerate

CCK-8 assay//SA--gal activity assay//Western blot

The miR-34a treated cells exhibited significant reduction in cell proliferation in comparison with the scrambled miR control group.miR-34a overexpression in ADSCs induced a significant decrease in expression of cyclins and CDKs.In contrast, miR-34a treatment induced the increased expression of the CDK inhibitors(P53,P21) in ADSCs.The number of SA-¦Â-gal-stained cells was markedly increased by miR-34a overexpression and recovered byco-treatment with anti-miR-34a, indicating the strong induction of cellular senescence by miR-34a.

--

Human

HL

cellular senescence

26,677,981

Gene

0

1

0

1

0

1

0

MIR377

494326

ncRNA

HSF

--

Aging

Accelerate

MTS assay//SA--gal activity assay//Western blot

Moreover, overexpression of miR-377 increased the SA-¦Â-gal-positive ratio and p16 expression while decreased HSF proliferation in young HSFs.

DNMT1//p53

Downregulation//Upregulation

qRT-PCR//Western blot

Computational miRNA target analysis from a miRNA database demonstrated that miR-377 had high homology with a sequence in the 3¡ä-UTR of human DNMT1 mRNA.DNMT1 mRNA and protein expression levels were significantly decreased by the miR-377 mimics and elevated by the miR-377 inhibitors.Of these three genes, p53 was further confirmed to be regulated by miR-377 using RT-qPCR and western blotting. Increased p53 mRNA,p53 and Rb expressions together with diminished phosphoryla- tion of Rb were induced by the miR-377 mimics and reverse changes were induced by the miR-377 inhibitors compared with that of the respective controls.

--

Human

HL

delay aging

28,277,545

Gene

0

1

0

1

0

1

0

MIR15B

406949

ncRNA

HDF

--

Aging

Prevent

qRT-PCR

We observed an increased expression at day 2 upon transfection of miR-15b inhibitors followed by a clear decrease of transcript levels (31-80%) at day 3 (IFN¦Ã) and day 4 (IL-1¦Á, IL-1¦Â, IL-6, IL-8, and VEGF).

SIRT4

Downregulation

Similarly, and again in accordance with the SIRT4 expression status,miR-15b levels were also decreased in UV- irradiated or gamma-irradiated senescent primary human dermal fibroblasts.In these experiments, miR-15b mimics dampened basal SIRT4 mRNA levels and inhibited senescence-associated upregulation of SIRT4 transcripts.Moreover, by employing luciferase assays we show that the 3¡¯UTR of human SIRT4 is a direct target of miR-15b, as miR-15b mimics significantly inhibited the relative luciferase activity of a transfected SIRT4 - 3¡¯-UTR construct, but not, when the SIRT4 ¨C 3¡¯-UTR was mutated within the seed sequence for miR-15b.

--

Human

HL

delay aging

26,959,556

Gene

0

1

0

MIR1292

100302138

ncRNA

Adipose stromal cell

--

Age-related osteoporosis

Accelerate

SA--gal activity assay//Western blot//qRT-PCR

qRT-PCR and western blot analyses also showed that lenti-1292-infected hADSCs expressed higher levels of the senescence markers P16 and P21 and lower expression of LMNB1 than control cells.Moreover, the number of SA-¦Â-gal-stained cells was increased after miR-1292 overexpression.

FZD4

Downregulation

Luciferase reporter assay//qRT-PCR//Western blot

Results showed that miR-1292 overexpression remarkably inhibited luciferase reporter activity from the vector containing the WT FZD4 3¡ä UTR, but not from the mutated FZD4 or those of other genes. Additionally, after miR-1292 overexpression, FZD4 was markedly down -regulated at the protein level, but not at mRNA level, Also, qRT-PCR results showed that the expression of miR-1292 was negatively correlated with FZD4 in 70 clinical bone samples.

Wnt-¦Â-catenin

Downregulation

Western blot

Western blotting studies revealed that miR-1292 overexpression decreased both FZD4 and ¦Â-catenin expression, indicating that Wnt/¦Â-catenin signaling was impaired.

Human

L

delay aging

30,574,422

Gene

0

1

0

1

0

1

0

CircPVT1

--

ncRNA

WI-38

--

Aging

Prevent

Cell morphological analysis//SA--gal activity assay//Western blot

Silencing CircPVT1 led to increased levels of senescence marker TP53(p53) and triggered a flattened and enlarged cell morphology accompanied by increased SA-¦Â gal activity.

let-7

Downregulation

qRT-PCR//SA-¦Â-gal activity assay//Cell morphological analysis//Western blot//Dual-Luciferase reporter assay

One of the microRNAs,let-7, was found to be the most highly enriched microRNA in the CircPVT1 pulldown.Importantly, silencing CircPVT1, which increases let-7 availability, significantly reversed this loss of P21 and TP53 expression observed with anti-let-7 alone.SA-¦Âgal activity was elevated in CircPVT1-silenced cells, while anti-let-7 had the opposite effect, reducing SA-¦Âgal activity and increasing proliferation; importantly, the proliferative phenotype seen after antagonizing let-7 was rescued when CircPVT1 was silenced. Although pre-let-7-transfected cells showed greater reduction in psiCHECK2-let-7 activity(RL/FL) (45% of control), silencing CircPVT1 also significantly lowered RL/FL activity (70% of control),indicating that CircPVT1 reduced the availability of functional let-7 to transcripts bearing a let-7 site.

--

Human

HL

delay aging

27,928,058

Gene

0

1

0

1

0

1

0

MEG3

55384

ncRNA

HUVEC

Blood

Aging

Prevent

FISH//Flow cytometry//Knockdown

The si-MEG3 + platelet group exhibited further elevated ROS production compared with the si-MEG3 group. Results of telomere length determination showed that telomere length was reduced by MEG3 silencing, and this reduction was enhanced by MEG3 silencing and platelets.

--

Human

L

delay aging

30,576,236

Gene

0

1

0

1

0

1

0

MIR15B

406949

ncRNA

--

Ovarian cancer

Accelerate

Flow cytometry//MTT assay//SA--gal activity assay

Cell proliferation, migration and invasion abilities in addition to cell proportion in the S phase in response to miR-15b mimic and siRNA LPAR3 significantly decreased while apoptosis and senescence, as well as cell proportion in G0/G1 phase significantly elevated (p < 0.05).

LPAR3

Downregulation

Dual-Luciferase reporter assay

The luciferase reporter exhibited that in comparison with the NC group, the luciferase activity of LPAR3 WT was profoundly reduced by miR 15b (p < 0.05), whereas that of the LPAR3 MUT was not changed.

PI3K-Akt

Downregulation

Western blot//qRT-PCR

In relative to the blank and NC groups, the miR-15b and Bax expression in ovarian cancer cells treated with miR 15b mimic dramatically enhanced (p < 0.05), but that of LPAR3 and Bcl 2, as well as the extent of PI3K and Akt phosphorylation, remarkably diminished (p < 0.05).

Human

HL

delay aging

31,140,597

Gene

0

1

0

1

0

1

0

MIR183

406959

ncRNA

HTM,HDF,HeLa

--

Aging

Accelerate

BrdU assay

Forced expression of miR-183 resulted in significant inhibition of cell proliferation in both primary HTM cells and HDFs but had no effect on the proliferation of HeLa cells.

--

Human

HL

delay aging

27,116,545

Gene

0

1

0