Epigenetic silencing of miR-126 contributes to tumor invasion and angiogenesis in colorectal cancer
ABSTRACT microRNAs (miRNAs) have been reported to play a crucial role in regulating a variety of genes pivotal for tumor metastasis. miR-126 is well known as one of the angiogenesis regulatory miRNAs. Recent studies have reported controversial roles of miR-126 in tumor progression. In this study, we sought to investigate the potential roles of miR-126 in colorectal cancer (CRC). By real-time PCR, miR-126 was shown to be downregulated in primary CRC tissues and cell lines. Restoration of miR-126 in CRC cells inhibited cell growth, migration and invasion. Using both in silico prediction and immunoblotting, we found that vascular endothelial growth factor (VEGF) was a target of miR-126. The interaction of miR-126 on the 3'UTR of VEGF mRNA was validated by luciferase reporter assay. Mechanistically, we found that the silencing of miR-126 was induced by promoter methyl-ation of its host gene, EGFL7. Treatment with 5-aza-CdR restored miR-126 expression and thereby led to a decline in VEGF expression. Functionally, due to suppression of VEGF, enhanced miR-126 expression inhibited tumor neovasculature triggered by CRC cells. In conclusion, our findings suggest that DNA methylation-induced silencing of miR-126 contributes, at least in part, to tumor invasion and angiogenesis in CRC, through upregulation of VEGF expression. miR-126 may be a potential target for the therapeutic strategy against CRC.
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ABSTRACT: miR-126 is an endothelial-specific microRNA essential for governing vascular integrity and angiogenesis. Its role in tumor angiogenesis of gastric cancer (GC) is unclear. This study aimed at determining the role of miR-126 in GC angiogenesis. Down-regulation of miR-126 was found to inversely correlate with an increased microvessel density (MVD) and vascular endothelial growth factor A (VEGF-A) expression in gastric cancer tissues. Bioinformatics analysis and luciferase reporter assay revealed that miR-126 directly targeted the 3'-untranslated region (3'-UTR) of VEGF-A mRNA. In addition, the restoration of miR-126 expression by lentivirus-miR-126 (Lenti-miR-126) transfection obviously reduced the expression of VEGF-A and the activition of its downstream genes, Akt, mTOR and Erk1/2 in gastric cancer cell lines SGC-7901, MKN-28 and MKN-45. In contrast, the down-regulation of miR-126 expression by lentivirus-anti-miR-126 (Lenti-anti-miR-126) transfection obviously up-regulated the expression of VEGF-A and its downstream signaling pathways. In vivo xenograft mice model experiments clarified the down-regulation of VEGF-A and MVD as well as inhibition of tumor growth by up-regulation of miR-126. Overall, the results from our study suggested that miR-126 could suppress tumor growth and tumor angiogenesis of GC through VEGF-A signaling, and it is a novel potential therapeutic target for GC.Oncotarget 11/2014; · 6.63 Impact Factor
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ABSTRACT: MicroRNAs (miRNAs) regulate the expression of hundreds of genes. However, identifying the critical targets within a miRNA-regulated gene network is challenging. One approach is to identify miRNAs that exert a context-dependent effect, followed by expression profiling to determine how specific targets contribute to this selective effect. In this study, we performed miRNA mimic screens in isogenic KRAS-Wild-type (WT) and KRAS-Mutant colorectal cancer (CRC) cell lines to identify miRNAs selectively targeting KRAS-Mutant cells. One of the miRNAs we identified as a selective inhibitor of the survival of multiple KRAS-Mutant CRC lines was miR-126. In KRAS-Mutant cells, miR-126 over-expression increased the G1 compartment, inhibited clonogenicity and tumorigenicity, while exerting no effect on KRAS-WT cells. Unexpectedly, the miR-126-regulated transcriptome of KRAS-WT and KRAS-Mutant cells showed no significant differences. However, by analyzing the overlap between miR-126 targets with the synthetic lethal genes identified by RNAi in KRAS-Mutant cells, we identified and validated a subset of miR-126-regulated genes selectively required for the survival and clonogenicity of KRAS-Mutant cells. Our strategy therefore identified critical target genes within the miR-126-regulated gene network. We propose that the selective effect of miR-126 on KRAS-Mutant cells could be utilized for the development of targeted therapy for KRAS mutant tumors.Oncotarget 07/2014; 5(17). · 6.63 Impact Factor
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ABSTRACT: Silencing of protein‑coding tumor suppressor genes (TSGs) by CpG island hypermethylation is a common occurrence in gastric cancer (GC). Here, we examine if tumor suppressor microRNAs (miRNAs) are silenced in a similar manner. Real‑time quantitative PCR (RTQ‑PCR) was employed to investigate the expression level of four candidate miRNAs in GC tissues (n=30) and cell lines. Basing on RTQ‑PCR results and bioinformatics approach, miR‑9 was chosen for further study on epigenetic regulation. Bisulfite genomic sequencing PCR (BSP) was performed to assess the methylation status of miR‑9 in GC tissues. In both GC cell lines and animal models, demethylation was performed either by treatment with 5‑aza‑2'‑deoxycytidine (5‑AZA‑CdR) or by siRNA targeting DNMT1. We also analyzed the relationship between miRNAs and several clinicopathological features. Candidate miRNAs (miR‑9, miR‑433, miR‑19b, and miR‑370) were found strongly downregulated in GC tissues and cell lines. Their expression was increased following 5‑AZA‑CdR treatment. CpG island methylation of miR‑9 was significantly higher in GC tissues compared to normal controls. After two demethylation treatments, miR‑9 methylation degree was significantly decreased and miR‑9 expression was ob-viously restored in GC cells and animal models. Deregulation of miR‑9 was positively correlated with tumor lesion size. Three other miRNAs, miR‑19b, miR‑433, and miR‑370 were assοciated with lymph node metastasis, decreased curvature, and poorly differentiated carcinoma. miR‑19b and miR‑433 were positively correlated with male gender. Of four candidate miRNAs downregulated in GC, miR‑9 is epigenetically regulated by DNA methylation both in vitro and in vivo.International Journal of Oncology 09/2014; 45(6). DOI:10.3892/ijo.2014.2667 · 2.77 Impact Factor