Study Of The Role And Mechanism Of MiR-126in Colorectal Cancer By Targeting CXCR4

Background:Colorectal cancer (CRC) is the third most common malignancy and the third leading cause of cancer-related mortality among men and women in the United States, accounting for9%of all cancer deaths in the Western world. Despite the improvement of multimodal anticancer strategies in recent years, the prognosis of advanced CRC is very poor, with5-year survival rates for stage III and stage IV colon cancer of65.4%and12.8%, respectively. Thus, it is critically important to further explore the molecular pathogenesis of CRC, which may provide new targets for therapeutic efficacy and clinical outcome.MicroRNAs (miRNAs) are a type of non-coding, endogenous, small RNAs with a length of approximately23nucleotides that can regulate gene expression via complementary base pairing with the3'untranslated region (3'-UTR) of target mRNAs, resulting in translational repression or mRNA degradation. MiRNAs are known to mediate diverse biological processes, such as cell proliferation, differentiation, apoptosis and development. Recent reports have indicated that some miRNAs are expressed abnormally in a wide range of malignancies and involved in cancer development, acting as as tumor suppressors as well as oncogenes. Recent evidence shows that altered microRNA-126(miR-126) expression is implicated in the progression of CRC. However, the exact roles and mechanisms of miR-126in CRC remain unclear. Thus, this study was to investigate the roles of miR-126in CRC and to clarify miR-126-mediated mechanisms in CRC.Part one:The expression analysis of microRNA-126in colon and its effect on biological properties in colorectal cancer cellsObjectives:To investigate the expression of miR-126in colorectal cancer (CRC), adjacent nontumorous tissues and CRC cells and to clarify the roles of miR-126in CRC cells.Methods:MiR-126expression in92pairs of CRC and adjacent nontumorous tissues and4human CRC cell lines (SW480, SW620, HT-29, and HCT-116) was examined using quantitative real-time PCR (qRT-PCR). Then, the biological properties of miR-126in CRC cells in vitro were examined by applying Cell Counting Kit8(CCK8), cell cycle, cell apoptosis, and transwell assays. Results:MiR-126expression was lower in CRC tissues and CRC cells than in adjacent nontumorous tissues. MiR-126overexpression inhibited cell proliferation, migration, and invasion of CRC cells by CCK8and transwell assay. Moreover, miR-126mimic induced G0/G1phase arrest, but did not affect the apoptosis of CRC cells by cell apoptosis assay.Conclusion:MiR-126was downregulated in CRC. And miR-126functions as a tumor suppressor by suppressing cell proliferation, migration, and invasion and induced cell arrest in the G0/G1phase of CRC cells.Part two:The possible mechanisms of microRNA-126-mediated tumor suppression in colorectal cancer cellsObjectives:To investigate the relationship between miR-126and CXC chemokine receptor4(CXCR4) and analyze the mechanisms of miR-126-mediated tumor suppression in CRC cells.Methods:The targets of miR-126were predicted by using bioinformatic analysis tools and proved by luciferase reporter assay. The effects of altered miR-126expression on CXCR4mRNA and protein were detected by using quantitative real-time PCR (qRT-PCR) and Western blot. Last, the AKT and ERK1/2signaling pathways of miR-126-mediated in CRC cells were assessed by Western blot.Results:We applied MicroCosm Targets and PicTar to predict putative binding sites of miR-126and found that the binding sites of miR-126are located at bases239-245nt of the CXCR43'-UTR, and CXCR4is a target of miR-126by using luciferase reporter assay. MiR-126overexpression reduced CXCR4protein level, while miR-126inhibitor enhanced CXCR4protein expression by Western blot analysis. CXCR4mRNA level remained unaltered with regard to miR-126level. Furthermore, miR-126overexpression led to decreased levels of phosphorylated AKT and ERK1/2, while transfection with miR-126inhibitor elevated the expression of phosphorylated AKT and ERK1/2. However, there was no difference in the levels of total AKT and ERK1/2with regard to miR-126.Conclusion:CXCR4is a target of miR-126. MiR-126negatively regulates CXCR4expression at the post-transcriptional level. Moreover, miR-126-mediated tumor suppression may be partly dependent on AKT and ERK1/2signaling pathways by targeting CXCR4expression. Part three:The association of microRNA-126expression and poor prognosis in colorectal cancerObjectives:To investigate the association between abnormal expression of miR-126and CXCR4expression and clinicopathological features of CRC patients and and to further study whether miR-126has any prognostic impact in patients with CRC.Methods:CXCR4mRNA expression in92pairs of CRC and adjacent nontumorous tissues was examined using quantitative real-time PCR (qRT-PCR), and CXCR4protein expression was assessed by immunohistochemistry (IHC) and Western blot. The correlation between miR-126and CXCR4protein expression and clinicopathological features was determined. Moreover, the association between miR-126and CXCR4protein expression and overall survival rate was also analyzed.Results:The expression of CXCR4mRNA was higher in CRC tissues than adjacent nontumorous tissues. IHC and Western blot also detected high expression of CXCR4protein in CRC tissues. An inverse correlation was observed between miR-126and CXCR4protein expression in CRC tissues. Moreover, low miR-126and high CXCR4protein expression was associated with distant metastasis and clinical TNM stage. Poor survival of CRC patients was also found to be strongly associated with a reduced level of miR-126expression and concomitant upregulation of CXCR4expression. And miR-126was identified as an independent prognostic indicator for overall survival of CRC patients.Conclusion:There was an inverse correlation between miR-126and CXCR4protein expression in CRC tissues. Moreover, miR-126was an independent prognostic predictor in CRC patients.