A Study On DNA Methylation And Biological Features Of MicroRNA-615 In Colorectal Cancer

[Background]MicroRNAs (miRNAs) have been suggested to play a critical role in tumor development. Recent studies showed about half of the human miRNA genes are related to CpG Islands (CGI) and epigenetic changes such as DNA methylation could regulate miRNA expression. In this study, we aimed at investigating the epigenetic regulation of miR-615 and its role in colorectal carcinogenesis.Previously on this study, the positive methylation rate of MIR615 in 46 CRC tissues was found significantly higher than that in paired normal colonic mucosa (39.1% versus 15.2%, p=0.003) while the expression of miR-615-3p was lower in CRC tissues than that in paired normal colonic mucosa (p=0.047). CRC tissues with positive MIR615 methylation showed a significantly lower expression of miR-615-3p than those with negative status (p=0.027). Methylation status of MIR615 was significantly correlated with patients’3-year disease-free survival (DFS) but not overall survival (OS). Patients with positive MIR615 methylation demonstrated a poor 3-year DFS than those were negative (60.2% versus 80.3%, p=0.005). Both univariate and multivariate analysis by Cox proportional hazards regression indicated that presence of perineural invasion and positive methylation status of MIR615 were two independent prognostic factors for stage Ⅱ/Ⅲ CRC patients.[Material and Methods]Colon cancer cell lines (SW480, SW620 and LoVo) were then investigated for MIR615 methylation level by quantitative methylation-specific PCR (qMSP) and expression by quantitative real-time reverse transcription-PCR (qRT-PCR) before and after 5-aza-2’-deoxycitidine (5-aza-dC) treatment to see whether demethylation interference can influence miR-615-3p and miR-615-5p expression. The mimics for miR-615-3p and miR-615-5p were transfected to cell line SW480 and LoVo to look at the potential biological function of miR-615-3p and miR-615-5p on cancer cell proliferation, migration and invasion. The database of computationally predicted human miRNA targets were to predict the candidate genes for miR-615-3p. KEGG (Kyoto encyclopedia of genes and genomes) pathway and Gene Ontology analysis were used to determine the possible candidate genes for miR-615-3p. Gene MEF2A showed the great possibility of target gene for miR-615-3p after the analysis. The mimics for miR-615-3p were transfected to cell line SW480 and LoVo to observe the mRNA expression of MEF2A. The mRNA expression of MEF2A in 27 pairs of CRC tissues and paired normal colonic mucosa was detected. The correlation between mRNA expression of MEF2A and miR-615-3p expression in 27 pairs of CRC tissues and paired normal colonic mucosa was analyzed.[Results]MIR615 was found embedded within a CpG Island using CpG Island Searcher program.5-aza-dC treatment increased both miR-615-3p [2.97-fold, 1.53-fold and 3.85-fold respectively (p<0.001)] and miR-615-5p [9.10-fold,4.99-fold and 9.93-fold respectively (p<0.001)] expression in SW480, SW620 and LoVo cell lines. Transfection of miR-615-3p and miR-615-5p mimics both inhibited cell growth, migration and invasion of SW480 and LoVo cell lines. Gene MEF2A showed the great possibility of target gene for miR-615-3p after the analysis. Overexpression of miR-615-3p down-regulated the mRNA expression of MEF2A in SW480 and LoVo cell lines (p<0.001, p<0.001). The negative correlation was observed between mRNA expression of MEF2A and miR-615-3p expression in 27 pairs of CRC tissues and paired normal colonic mucosa (r=-0.541, p<0.001).[Conclusion]Demethylation treatment of CRC cell lines increased the expression level of miR-615-3p and miR-615-5p. Aberrant methylation of CpG Island for MIR615 could regulate the expression of miR-615-3p and miR-615-5p. Both miR-615-3p and miR-615-5p act as potential tumor suppressors which inhibit proliferation, migration and invasion in vitro for CRC cell lines. MEF2A could be the possible target gene for miR-615-3p.