MicroRNA-135b Promotes Cancer Progression by Acting as a Downstream Effector of Oncogenic Pathways in Colon Cancer

Summary MicroRNA deregulation is frequent in human colorectal cancers (CRCs), but little is known as to whether it represents a bystander event or actually drives tumor progression in vivo. We show that miR-135b overexpression is triggered in mice and humans by APC loss, PTEN/PI3K pathway deregulation, and SRC overexpression and promotes tumor transformation and progression. We show that miR-135b upregulation is common in sporadic and inflammatory bowel disease-associated human CRCs and correlates with tumor stage and poor clinical outcome. Inhibition of miR-135b in CRC mouse models reduces tumor growth by controlling genes involved in proliferation, invasion, and apoptosis. We identify miR-135b as a key downsteam effector of oncogenic pathways and a potential target for CRC treatment.


SUPPLEMENTAL DATA
List of miRs deregulated in cancer compared to normal tissues in the AOM/DSS mice. p value < 0.001. Deregulation greater than two-fold. Red indicates miRs that are up-regulated, green those that are down-regulated.    Hsa-miR-135b expression was assessed by real time PCR. The expression of miR-135b was normalized to that of RNU48, while that of other genes to GAPDH. Bars represent the mean and SD of 3 experiments, p values are reported within the figure.
(A) APC mRNA expression in SW480 cells transfected with an APC-encoding plasmid.
(B) APC mRNA expression in NCM460 cells transfected with APC siRNA.
(E) miR-135b expression after transfection with selected siRNA targeting transcription factors downstream of the APC/-catenin axis.
(F) MiR-135b expression assessed by RT-PCR in DLD-1 isogenic human CRC cells with or without mutation of the PI3K after exposure to 10% or 0.5% of fetal bovine serum (FBS).
(I) miR-135b promoter activation (blue bars) and ratio between TOPFLASH and FOPFLASH (red bars) luciferase activity following treatment with LiCl in presence/absence of FOXO3a modulation.  (A) miR-135b expression assessed by RT-PCR in isogenic human CRC cells transfected with pre-miR-135b, LNA anti-miR-135b and relative controls. The expression of miR-135b was normalized to that of RNU48.
(B) Isogenic PI3K WT and mutant DLD-1 cells were transfected with pre-miR-135b, LNA anti-miR-135b or relative controls. Cell viability was measured at selected time points. p values are reported in Table S5.
(C) Isogenic PI3K mutant and WT cells over-expressing miR-135b or anti-miR-135b were plated in soft agar and grown in low FBS conditions. Colonies greater than 2 mm in size were counted and quantitated.  The expression of miR-135b was normalized to that of RNU48 or SNU234 for human and mice experiments respectively. Bars represent the mean and SD of 3 mice, p value <0.001.
(A) Human CRC cell lines were treated with a probe anti-miR-135b (miR-135b-AMO) or a scrambled probe (scrambled-AMO) and miR-135b expression assessed by RT-PCR.
(B) 8 week-old BL/6 wild type mice were treated with miR-135b-AMO. ISH was performed using an anti-anti-miR-135b (a specific LNA probe designed to recognize the 135b-AMO).
(C) Expression of miR-21, miR-155, miR-221 and let7-d was evaluated by RT-PCR to test 135b-AMO off-target effects.  (C) Morphology of lungs, livers, kidneys and spleens from miR-135b-AMO and scrambled-AMO treated mice were compared by H&E. GFP was assessed by Immunohistochemistry in xeno-transplanted organoids 8 weeks after transplantation.

Real time PCR for mature miRNAs and genes
Total RNA was isolated using Trizol (Invitrogen, Carlsbad, CA SRC-MEFs were infected with lentiviruses expressing anti-miR-135b hairpins or empty controls. MEFs were counted and plated as described above and GFP was monitored over time with the use of EVOS microscopy (LifeTechnology). Total colonies were counted after 4 weeks.

Tube Formation Assay
NCM 460 cells were transfected with pre-miR-135b or pre-miR-control under starvation.
16 hr after transfection, media from both experiments was collected and used to culture HUVEC Matrigel (BD PharMingen; 12.9 mg/mL)-coated LabTek (55,000 cells per chamber). Results are expressed as number of tube-like structures per field (magnification, ×200). Mice were treated as follows:

RNAs from
AOM + DSS + Scrambled-AMO (Scrambled-AMO group) (n=8) C57BL/6 8 week-old mice were treated with AOM i.p. at day 0 and received 2.5% DSS water according to the above mentioned schedule. Anti-miR-control was given twice a week for the entire treatment (

Rapid Apc inactivation in Apc fl/fl mice
To induce recombination, mice were given daily intraperitoneal injections of βnapthoflavone (80 mg/kg) as previously described (Sansom et al., 2004). At each time point, mice were killed and intestines removed and flushed with water. Intestines were dissected as follows: The proximal 7 cm was mounted en face, fixed overnight in methacarn (methanol, chloroform, and acetic acid; 4:2:1 ratio), and paraffin embedded.
The following 3 cm was opened and placed into RNA later (Sigma), ensuring that all mesentery and Peyers patches were removed. The following 5 cm was divided into 1-cm lengths, bundled using surgical tape, and then fixed in 4% formaldehyde at 4°C for no more than 24 h before processing. Samples for Western analysis, frozen sections, and electron microscopy were then taken from the next 5 cm of intestine, and the remainder was fixed in methacarn. Apoptosis and mitotic index were scored from hematoxylin-andeosin-stained sections as previously described (Sansom et al., 2004). Apoptosis was independently confirmed by immunohistochemical staining with an antibody against active caspase 3 (1:750; R&D systems). For BrdU labeling, mice were injected with 0.25 mL of BrdU (Amersham), and staining was performed using an anti-BrdU antibody conjugate (Roche) at 1 part in 50.

Histological Analysis
Colons were examined using 4 μm thick, 200 μm step serial sections stained with hematoxylin and eosin. For TUNEL assay, an In Situ Cell Death Kit (Roche, Indianapolis, Indiana) was used according to the manufacturer's recommendations. MicroRNA detection was performed on colon cancer tissues from mice intestines or human colon cancer sections by in situ hybridization (ISH) as previously described (Nuovo 2010). The negative controls included omission of the probe and the use of a scrambled LNA probe.
Immunohistochemistry for Ki-67 was performed as previously described at the OSU comparative pathology core facility.