Up-frameshift Protein 1 Promotes Tumor Progression by Regulating Apoptosis and Epithelial–Mesenchymal Transition of Colorectal Cancer
Colorectal cancer (CRC) is the third frequent malignancy and is the second leading cause of cancer-related deaths worldwide. Incident rates of CRC rose by 2.2% annually overall. Although screening and treatments of CRC get advanced recently, majority of patients hold a poor prognosis. Therefore, better understanding of potential prognosis biomarkers and therapeutic targets is needed to improve survival of CRC patients. Up-frameshift protein 1 (UPF1), located at chromosome 19p13.2-p13.11, is a highly conserved and ubiquitously expressed phosphoprotein with RNA/DNA-dependent ATPase and RNA helicase activity. Depending on its ATPase and helicase activities, , UPF1 serves as the core regulator of nonsense-mediated mRNA decay machinery, which is a RNA surveillance pathway protecting cells from damage by premature termination codons. Also, UPF1 is involved in cell proliferation and differentiation. , In addition, UPF1 is required for S phase progression and genome stability, which is essential for DNA damage repair. Previous studies have clarified the exact activities of UPF1 in hepatocellular cancer and gastric cancer, however, UPF1 exact biological activities in human CRC are poorly understood. DNA damage is the well-known trigger of the intrinsic apoptosis pathway. Signaling to intrinsic apoptosis due to DNA damage stimuli the release of cytochrome C and activate BCL-2 family, promoting the formation of apoptosome. , Then, procaspase 9 is activated, which in turn activates the executioner apoptosis cleaved caspase 3, leading to the death of cells. Double strand breaks (DSB) are wildly recognized to be among the most deadly forms of DNA damage, causing genomic unstability. H2AX is considered to be the hallmark of DNA damage and DSB, its phosphorylation is tightly linked to DNA damage repairs. According to reports, the expression of H2AX is increased in UPF1-depleted cells. These observations arise our interest to explore whether and how UPF1 and H2AX are involved in tumorigenesis of CRC and the underlying mechanism driving the relationships with apoptosis. In this study, we demonstrated that UPF1 was significantly upregulated in CRC tissues compared with adjacent normal tissues. Subsequently, we investigated the contribution of UPF1 to the proliferation and metastasis of CRC as well as its effect on DNA damage and apoptosis, which provides a new sight for clinical diagnosis and treatment of CRC. 

Colorectal Clinical Specimens and Cell Lines Forty-eight pairs of CRC specimens and paired adjacent noncancer tissues were collected from patients of The First Affiliated Hospital of Shantou University Medical College. Tumor staging relies on eighth American Joint Committee on Cancer Tumor-node-metastasis classification (AJCC TNM-8). All the patients who participated in this study had signed the written informed consent voluntarily. The present study was approved by the Medical Ethics Committee of The First Affiliated Hospital of Shantou University Medical College. The FHC human normal colorectal epithelial cell line and 4 human CRC cell lines (SW480, SW620, RKO, Caco-2) were cultured with DMEM supplemented with 10% fetal bovine serum (Gibco, Australia origin) in a water-saturated environment at 37 °C with 5% CO 2 . All the cell lines (FHC, SW480, SW620, RKO, Caco-2) were donated by General surgery laboratory of Southern Medical University. Immunohistochemistry Immunohistochemical staining of 48 pairs of CRC tissues and paired normal tissues was performed on 4 μm thick paraffin sections. All the procedures were referred to manufacturer's protocol using an UltraSensitiveTM S-P kit and DAB (Maixinbio, China). Antibody UPF1(dilution of 1:10000, ab109363, abcam) was applied. Cytoplasm stained with brownish yellow is considered positive. The staining paraffin sections were imaged with microscope (200× magnification). Image J software was used to evaluate the immunohistochemistry (IHC) staining. The percent positively stained areas were scored as “1” (0-25%), “2” (26-50%), and “3” (≥50%). The staining intensity was scored as “0” (no staining), “1” (weak), “2” (moderate), and “3” (strong). Then we calculated the sum of the 2 scores. The total score ≤3 was defined as negative expression, while the total score ≥4 was defined as positive expression. All specimens were evaluated by 2 pathologists who were blinded to the patient identity and clinical outcome. Quantitative Real-Time Polymerase Chain Reaction Total RNA was extracted from fresh tissue using EasyPure RNA Kit (TransGen) and was reverse-transcribed into cDNA using a PrimeScript RT Master Mix (TaKaRa). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed using SYBR Premix Ex Taq II Kit (TaKaRa) on CFX Connect. β-actin was used as an internal control. The relative expression was evaluated using 2 −ΔΔct method. The primers used are as follows: β-actin (5’-TGGCACCCAGCACAATGAA-3’; 5’-CTAAGTCATAGTCCGCCTAGAAGCA-3’); UPF1 (5’-AGAGCCTCATGCAGTTCAGCAA-3’; 5’-GGCATCATACATGGCTGTGGTC-3’). Plasmid Constructions and Transfection UPF1-shRNAs were designed and synthesized by GeneCopoeia. The sequence of shRNAs used are as follows UPF1-shRNA #1: 5’-GCAGCCACATTGTAAATCACC-3’; UPF1-shRNA #2: 5’-GCGTGGTTTACTGTAATACCA-3’; UPF1-shRNA #3: 5’-CCTATTACACGAAGGACCTCC-3’. CRC cell lines SW620 and RKO were transfected using Lipofectamine 3000 Transfection Reagent (Thermo Fisher). At 48 h after transfection, qRT-PCR and western blotting were used to confirm the expression of UPF1. Cell Proliferation Assay CCK-8 assay was used to examine cell proliferation. SW620 and RKO cell lines with UPF1 knockdown were seeded on the 96-well plates at a concentration of 3 × 10 3 of RKO while 5 × 10 3 of SW620 per well with 100 μl DMEM containing 10% CCK-8 solution (CCK-8, Dojindo). Then, each well was cultured for 24, 48, 72, and 96 h. Cells with CCK-8 reagent were incubated for 4 h at 37 °C. Finally, the absorbance value (OD) of 450 nm was read on the microplate reader (Bio-Rad). EdU staining was also used to assess cell proliferation. CRC cells seeded in each well of 24-well plates at a density of 4 × 10 4 cells/well of RKO while 6 × 10 4 cells/well of SW620 were stained using Meilun EdU cell proliferation Kit with Alexa Fluor 488 (Meilun, Dalian) referring to manufacturer's protocol. Lastly, the cells were imaged with inverted microscope (100× magnification). Transwell Assay The migration and invasion of cells were assessed using transwell chamber (0.8 mm) with Matrigel-coated or not. Transfected RKO cells (4 × 10 4 ) and SW620 cells (6 × 10 4 ) were separately seeded in upper chamber with 100 μl serum-free medium. The lower chamber was filled with 500 μl medium containing 20% FBS. After 48 h, cells trapped in the chamber membrane surface were fixed with 4% paraformaldehyde for 15 min, stained using 0.1% crystal violet for 15 min and imaged with microscope (400× magnification). Migrated and invaded cells of each sample were counted in 5 random fields for statistical analysis. Flow Cytometric Analyses Transfected RKO cells and SW620 cells were collected 48 h after transfection and then stained using cell cycle detection kit (MultiSciences) following the manufacturer's instructions. The percentage of cells in the cell cycle stages was analyzed by a FACSCanto II flow cytometer (BD Biosciences). For the cell apoptosis assays, transfected cells were analyzed using Annexin V-FITC/PI apoptosis detection kit (4A Bio) according to the manufacturer's instructions, cell apoptosis rate was then evaluated by FACSCanto II flow cytometer. Western Blotting Total proteins in cells were extracted by radio-immunoprecipitation assay buffer (RIPA) and the proteins concentration was measured using the bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific). The targeted proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (5% stacking and 12% separating gels) and transferred onto PVDF membranes which were blocked with 5% bovine serum albumin (BSA) solution for 1 h. Then the polyvinylidene fluoride (PVDF) membranes with targeted proteins were incubated with primary antibody including UPF1 (dilution of 1:10000, ab109363, abcam), β-actin (dilution of 1:1000, 4ab000001, 4A Biotech), E-cadherin (dilution of 1:1000, #3195, cell signaling), Vimentin (dilution of 1:1000, #5741, cell signaling), H2AX (dilution of 1:1000, #7631, cell signaling), Bax(dilution of 1:1000, #5023, cell signaling), Bcl-2 (dilution of 1:1000, Ab32124, abcam), Caspase3 (dilution of 1:1000, #14220, cell signaling), Cleaved Caspase3 (dilution of 1:1000, #9661, cell signaling), and paired secondary antibody goat anti-rabbit/mouse IgG H&L (HRP) (dilution of 1:2000, SA00001-1/ SA00001-2, Proteintech). Finally, the proteins bands were detected using ECL chemiluminescence system. Statistical Analysis All experiments were repeated at least 3 times and the data were expressed as the means ± standard deviation. The data were evaluated using Student's t -test and X 2 -test. And P < .05 was considered to be significant. Statistical graphs were performed using GraphPad Prism software. 

Forty-eight pairs of CRC specimens and paired adjacent noncancer tissues were collected from patients of The First Affiliated Hospital of Shantou University Medical College. Tumor staging relies on eighth American Joint Committee on Cancer Tumor-node-metastasis classification (AJCC TNM-8). All the patients who participated in this study had signed the written informed consent voluntarily. The present study was approved by the Medical Ethics Committee of The First Affiliated Hospital of Shantou University Medical College. The FHC human normal colorectal epithelial cell line and 4 human CRC cell lines (SW480, SW620, RKO, Caco-2) were cultured with DMEM supplemented with 10% fetal bovine serum (Gibco, Australia origin) in a water-saturated environment at 37 °C with 5% CO 2 . All the cell lines (FHC, SW480, SW620, RKO, Caco-2) were donated by General surgery laboratory of Southern Medical University. 

Immunohistochemical staining of 48 pairs of CRC tissues and paired normal tissues was performed on 4 μm thick paraffin sections. All the procedures were referred to manufacturer's protocol using an UltraSensitiveTM S-P kit and DAB (Maixinbio, China). Antibody UPF1(dilution of 1:10000, ab109363, abcam) was applied. Cytoplasm stained with brownish yellow is considered positive. The staining paraffin sections were imaged with microscope (200× magnification). Image J software was used to evaluate the immunohistochemistry (IHC) staining. The percent positively stained areas were scored as “1” (0-25%), “2” (26-50%), and “3” (≥50%). The staining intensity was scored as “0” (no staining), “1” (weak), “2” (moderate), and “3” (strong). Then we calculated the sum of the 2 scores. The total score ≤3 was defined as negative expression, while the total score ≥4 was defined as positive expression. All specimens were evaluated by 2 pathologists who were blinded to the patient identity and clinical outcome. 

Total RNA was extracted from fresh tissue using EasyPure RNA Kit (TransGen) and was reverse-transcribed into cDNA using a PrimeScript RT Master Mix (TaKaRa). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed using SYBR Premix Ex Taq II Kit (TaKaRa) on CFX Connect. β-actin was used as an internal control. The relative expression was evaluated using 2 −ΔΔct method. The primers used are as follows: β-actin (5’-TGGCACCCAGCACAATGAA-3’; 5’-CTAAGTCATAGTCCGCCTAGAAGCA-3’); UPF1 (5’-AGAGCCTCATGCAGTTCAGCAA-3’; 5’-GGCATCATACATGGCTGTGGTC-3’). 

UPF1-shRNAs were designed and synthesized by GeneCopoeia. The sequence of shRNAs used are as follows UPF1-shRNA #1: 5’-GCAGCCACATTGTAAATCACC-3’; UPF1-shRNA #2: 5’-GCGTGGTTTACTGTAATACCA-3’; UPF1-shRNA #3: 5’-CCTATTACACGAAGGACCTCC-3’. CRC cell lines SW620 and RKO were transfected using Lipofectamine 3000 Transfection Reagent (Thermo Fisher). At 48 h after transfection, qRT-PCR and western blotting were used to confirm the expression of UPF1. 

CCK-8 assay was used to examine cell proliferation. SW620 and RKO cell lines with UPF1 knockdown were seeded on the 96-well plates at a concentration of 3 × 10 3 of RKO while 5 × 10 3 of SW620 per well with 100 μl DMEM containing 10% CCK-8 solution (CCK-8, Dojindo). Then, each well was cultured for 24, 48, 72, and 96 h. Cells with CCK-8 reagent were incubated for 4 h at 37 °C. Finally, the absorbance value (OD) of 450 nm was read on the microplate reader (Bio-Rad). EdU staining was also used to assess cell proliferation. CRC cells seeded in each well of 24-well plates at a density of 4 × 10 4 cells/well of RKO while 6 × 10 4 cells/well of SW620 were stained using Meilun EdU cell proliferation Kit with Alexa Fluor 488 (Meilun, Dalian) referring to manufacturer's protocol. Lastly, the cells were imaged with inverted microscope (100× magnification). 

The migration and invasion of cells were assessed using transwell chamber (0.8 mm) with Matrigel-coated or not. Transfected RKO cells (4 × 10 4 ) and SW620 cells (6 × 10 4 ) were separately seeded in upper chamber with 100 μl serum-free medium. The lower chamber was filled with 500 μl medium containing 20% FBS. After 48 h, cells trapped in the chamber membrane surface were fixed with 4% paraformaldehyde for 15 min, stained using 0.1% crystal violet for 15 min and imaged with microscope (400× magnification). Migrated and invaded cells of each sample were counted in 5 random fields for statistical analysis. 

Transfected RKO cells and SW620 cells were collected 48 h after transfection and then stained using cell cycle detection kit (MultiSciences) following the manufacturer's instructions. The percentage of cells in the cell cycle stages was analyzed by a FACSCanto II flow cytometer (BD Biosciences). For the cell apoptosis assays, transfected cells were analyzed using Annexin V-FITC/PI apoptosis detection kit (4A Bio) according to the manufacturer's instructions, cell apoptosis rate was then evaluated by FACSCanto II flow cytometer. 

Total proteins in cells were extracted by radio-immunoprecipitation assay buffer (RIPA) and the proteins concentration was measured using the bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific). The targeted proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (5% stacking and 12% separating gels) and transferred onto PVDF membranes which were blocked with 5% bovine serum albumin (BSA) solution for 1 h. Then the polyvinylidene fluoride (PVDF) membranes with targeted proteins were incubated with primary antibody including UPF1 (dilution of 1:10000, ab109363, abcam), β-actin (dilution of 1:1000, 4ab000001, 4A Biotech), E-cadherin (dilution of 1:1000, #3195, cell signaling), Vimentin (dilution of 1:1000, #5741, cell signaling), H2AX (dilution of 1:1000, #7631, cell signaling), Bax(dilution of 1:1000, #5023, cell signaling), Bcl-2 (dilution of 1:1000, Ab32124, abcam), Caspase3 (dilution of 1:1000, #14220, cell signaling), Cleaved Caspase3 (dilution of 1:1000, #9661, cell signaling), and paired secondary antibody goat anti-rabbit/mouse IgG H&L (HRP) (dilution of 1:2000, SA00001-1/ SA00001-2, Proteintech). Finally, the proteins bands were detected using ECL chemiluminescence system. 

All experiments were repeated at least 3 times and the data were expressed as the means ± standard deviation. The data were evaluated using Student's t -test and X 2 -test. And P < .05 was considered to be significant. Statistical graphs were performed using GraphPad Prism software. 

UPF1 Expression is Significantly Upregulated in 
CRC and Correlates with Patients’ Prognosis To evaluate the expression and clinical significances of UPF1 in CRC, we first measured its expression level in 48 CRC patients using IHC staining. The results showed that UPF1 was significantly upregulated in CRC tissues compared to adjacent normal tissues ( ). qRT-PCR was further performed to examine the expression of UPF1 in 48 CRC patients. As shown in , UPF1 expression was higher in CRC tissues than in adjacent normal mucosa. Next, we separated these patients into UPF1 high (n = 23) and low (n = 25) group according to median value. Clinical data showed that aberrant expression of UPF1 was positively associated with the positive lymph node metastasis and TMN stage ( , ). Then, we downloaded the data containing 597 patients from the TCGA-COAD dataset. Kaplan-Meier and log-rank test analyses showed that patients with higher expression of UPF1 had significantly shorter overall survival (OS) ( ). Knockdown of UPF1 Inhibits CRC Cell Proliferation 
and Cell Cycle Progression Similarly, qRT-PCR showed that UPF1 expression was higher in a group of CRC cell lines compared with FHC cell line, which is an immortalized normal human colorectal cell line ( ). Western blotting further proved the higher expression of UPF1 in CRC cell lines ( ), especially in SW620 and RKO. Therefore, these 2 cell lines were employed to confirm the oncogene role of UPF1 in vitro. To further investigate the biological function of UPF1, we knocked down UPF1 in SW620 and RKO cells by transfecting specific shRNA against UPF1. The results showed that the mRNA and protein levels were significantly reduced in 2 CRC cells transfected with sh-UPF1-1 and sh-UPF1-2 ( ). CCK8 and EdU proliferation assays revealed that silencing UPF1 inhibited cell proliferation relative to the negative control (NC) groups ( -D). In addition, the cell cycle assays showed that silencing of UPF1 increased the proportion of cells in S phase ( ). Knockdown of UPF1 Inhibits Cell Migration, 
Invasion, and EMT in CRC Cells To further examine the effects of UPF1 on tumor metastasis, we evaluated the biological activity of UPF1 in regulating migration and invasion through transwell assays. As shown in , silencing of UPF1 repressed the migration and invasion abilities of SW620 and RKO cells ( ). As EMT is well known for tumor metastasis, the protein level of EMT markers was evaluated by western blotting. Silencing of UPF1 increased the expression of epithelial marker E-cadherin, while repressed the expression of mesenchymal marker vimentin ( ). Taken together, our results revealed that knockdown of UPF1 inhibits cell migration, invasion, and EMT in CRC. Knockdown of UPF1 Promotes Apoptosis by Inducing DNA Damage in CRC According to the study by Azzalin and his colleagues, UPF1 was essential to DNA stability, and DNA damage might be triggered by silencing of UPF1. Previous studies had demonstrated that UPF1 was involved in apoptosis regulation, , however, whether UPF1 could regulate apoptosis through triggering DNA damage promotes us to explore the underlying mechanism in CRC cells. As shown, the expression of DNA damage marker H2AX was significantly upregulated when UPF1 was silenced in SW620 and RKO cells ( ). Then, apoptosis flow cytometry analysis (FACS) showed that UPF1-knockdown induced apoptosis in the 2 cells ( ). We also evaluated the expression of apoptosis markers in cells transfected with UPF1-shRNA. The results showed that knockdown of UPF1 increased the pro-apoptosis marker BAX protein expression while decreased the anti-apoptosis marker Bcl-2 protein expression. In addition, cleaved caspase 3 were upregulated in UPF1-knockdown CRC cells compared to control cells, while the protein levels of procaspase 3 were not significantly altered ( ). In addition, DNA mismatch repair (MMR) is also implicated in apoptosis after DNA damage. In order to explore the relationship between UPF1 and MMR proteins, we performed analysis using GEPIA database. As shown in Supplemental Figure S1 , expression of UPF1 was positively correlated with MMR proteins including MLH1, MSH2, MSH6, and PMS2 ( Supplemental Fig. S1 A-D ). Therefore, we concluded that knockdown of UPF1 caused damage to DNA, and consequently promoted apoptosis in CRC cells. 

To evaluate the expression and clinical significances of UPF1 in CRC, we first measured its expression level in 48 CRC patients using IHC staining. The results showed that UPF1 was significantly upregulated in CRC tissues compared to adjacent normal tissues ( ). qRT-PCR was further performed to examine the expression of UPF1 in 48 CRC patients. As shown in , UPF1 expression was higher in CRC tissues than in adjacent normal mucosa. Next, we separated these patients into UPF1 high (n = 23) and low (n = 25) group according to median value. Clinical data showed that aberrant expression of UPF1 was positively associated with the positive lymph node metastasis and TMN stage ( , ). Then, we downloaded the data containing 597 patients from the TCGA-COAD dataset. Kaplan-Meier and log-rank test analyses showed that patients with higher expression of UPF1 had significantly shorter overall survival (OS) ( ). 

Similarly, qRT-PCR showed that UPF1 expression was higher in a group of CRC cell lines compared with FHC cell line, which is an immortalized normal human colorectal cell line ( ). Western blotting further proved the higher expression of UPF1 in CRC cell lines ( ), especially in SW620 and RKO. Therefore, these 2 cell lines were employed to confirm the oncogene role of UPF1 in vitro. To further investigate the biological function of UPF1, we knocked down UPF1 in SW620 and RKO cells by transfecting specific shRNA against UPF1. The results showed that the mRNA and protein levels were significantly reduced in 2 CRC cells transfected with sh-UPF1-1 and sh-UPF1-2 ( ). CCK8 and EdU proliferation assays revealed that silencing UPF1 inhibited cell proliferation relative to the negative control (NC) groups ( -D). In addition, the cell cycle assays showed that silencing of UPF1 increased the proportion of cells in S phase ( ). 

To further examine the effects of UPF1 on tumor metastasis, we evaluated the biological activity of UPF1 in regulating migration and invasion through transwell assays. As shown in , silencing of UPF1 repressed the migration and invasion abilities of SW620 and RKO cells ( ). As EMT is well known for tumor metastasis, the protein level of EMT markers was evaluated by western blotting. Silencing of UPF1 increased the expression of epithelial marker E-cadherin, while repressed the expression of mesenchymal marker vimentin ( ). Taken together, our results revealed that knockdown of UPF1 inhibits cell migration, invasion, and EMT in CRC. 

According to the study by Azzalin and his colleagues, UPF1 was essential to DNA stability, and DNA damage might be triggered by silencing of UPF1. Previous studies had demonstrated that UPF1 was involved in apoptosis regulation, , however, whether UPF1 could regulate apoptosis through triggering DNA damage promotes us to explore the underlying mechanism in CRC cells. As shown, the expression of DNA damage marker H2AX was significantly upregulated when UPF1 was silenced in SW620 and RKO cells ( ). Then, apoptosis flow cytometry analysis (FACS) showed that UPF1-knockdown induced apoptosis in the 2 cells ( ). We also evaluated the expression of apoptosis markers in cells transfected with UPF1-shRNA. The results showed that knockdown of UPF1 increased the pro-apoptosis marker BAX protein expression while decreased the anti-apoptosis marker Bcl-2 protein expression. In addition, cleaved caspase 3 were upregulated in UPF1-knockdown CRC cells compared to control cells, while the protein levels of procaspase 3 were not significantly altered ( ). In addition, DNA mismatch repair (MMR) is also implicated in apoptosis after DNA damage. In order to explore the relationship between UPF1 and MMR proteins, we performed analysis using GEPIA database. As shown in Supplemental Figure S1 , expression of UPF1 was positively correlated with MMR proteins including MLH1, MSH2, MSH6, and PMS2 ( Supplemental Fig. S1 A-D ). Therefore, we concluded that knockdown of UPF1 caused damage to DNA, and consequently promoted apoptosis in CRC cells. 

Our study showed that UPF1 was upregulated in CRC tissues and cell line. Moreover, high expression promoted cell growth and metastasis of malignant CRC cells, which agreed with the clinicopathological parameter. The expression of UPF1 was associated closely with TMN stages and lymphatic penetration in CRC. Therefore, our data demonstrated that UPF1 has a potential to act as a prognostic biomarker for CRC. However, more samples should be included to determine the clinical value of UPF1 in CRC. EMT is recognized to be a crucial mechanism that plays an important role in cancer invasion and metastasis. In this process, cells which lose epithelial characteristics gain migratory and invasive properties. Our study elucidated that knockdown of UPF1 increased expression E-cadherin with reduced expression of Vimentin. These data suggested that UPF1 could activate process of EMT in CRC, leading to the enhanced metastasis. Tumor budding is a highly aggressive subpopulation of tumor cells with migratory and invasive capacities. According to researches of Gurzu et al., EMT was associated with the tumor-budding degree in CRC. Budding degree could be used to evaluate EMT in CRC. , However, whether UPF1 was correlated with tumor budding and the mechanism behind EMT are unclear, which should be further investigated in the following research. Previous studies have demonstrated that UPF1 was essential in various biological processes including embryonic and brain development. In Drosophila melanogaster, UPF1 loss-of-function suppressed cell growth and triggered apoptosis. However, dysfunction of UPF1 also contributed in tumorigenesis through regulating apoptosis. Actually, how UPF1 inducing apoptosis still remains unclear. As Azzalin and his colleagues mentioned, UPF1 was necessary to genome stability and UPF1-depletion might trigger DNA damage. , In our study, we found that UPF1 silencing induced DNA damage response and increased the proportion of cells in S phase. Intriguingly, apoptotic rate in UPF1-depleted cell was significantly increased. It is known that intrinsic apoptosis pathway signal could arise from DNA damage. Taken together, we concluded that UPF1-depletion regulated apoptosis through inducing DNA damage. However, more downstream functional experiments still determine to be clarified in the following research. Actually, the function of UPF1 is required for genome stability and is dual in tumorigenesis. Chang et al. clarified that UPF1 could suppress HCC invasion, migration, and proliferation through activating the TGF-β/Smad pathway by eliminating LncRNA SNHG6. Whereas, Shao and his colleagues demonstrated that UPF1 increased cell viability and promoted metastasis via enhancing the stability of Linc-00313 in glioma. Our results showed that knockdown of UPF1 suppressed the malignant biological behaviors of CRC cells via triggering DNA damage. It seems that UPF1 phosphorylated by different kinases lead to different DNA or RNA metabolism processes. On the one hand, phosphorylated by SMG-1, UPF1 recognizes targeted mRNA and then works with UPF2/UPF3 to trigger the decay of aberrant RNA. , On the other, modified by ATR phosphorylation, UPF1 is essential in DNA stability. Herein, we elucidated regulatory roles of UPF1 in CRC. However, whether DNA damage caused by UPF1 knockdown was induced by ATR phosphorylation in CRC cells should be further investigated in the future. 

Our study suggested that the upregulation of UPF1 in tumor tissues was significantly correlated with poor prognosis. Furthermore, UPF1 depletion was involved in CRC progression through activated apoptosis and EMT progression. All the results indicated that UPF1 played an important role in CRC carcinogenesis and may serve as a potential target for CRC diagnosis and therapy. 

sj-docx-1-tct-10.1177_15330338211064438 - Supplemental material for Up-frameshift Protein 1 Promotes Tumor Progression by Regulating Apoptosis and Epithelial–Mesenchymal Transition of Colorectal Cancer Click here for additional data file. Supplemental material, sj-docx-1-tct-10.1177_15330338211064438 for Up-frameshift Protein 1 Promotes Tumor Progression by Regulating Apoptosis and Epithelial–Mesenchymal Transition of Colorectal Cancer by Binlie Chen, Huaiming Wang, Danfeng Li, Xiaosheng Lin, Zhiyan Ma and Yongming Zeng in Technology in Cancer Research & Treatment 

