Correlations Between MiR-139-5p And The Biological Function Of Colon Cancer Cell Lines

Objective:Colon Cancer is a common malignant tumor of digestive system, the morbidity and mortality of Colon Cancer is increasing year by year, in western countries, the incidence of colon cancer is in the second place of world, while in our country, its incidence and mortality is located in the fourth. At present, although colon cancer has made some achievements in its early diagnosis and early treatment, the prognosis and5years survival rate are still very low.This study observed the influence of the expression of miR-139-5p in colon cancer cell proliferation, apoptosis, cell cycle, invasion and migration. Preliminary discussing the role of miR-139-5p in development of colon cancer. It will provide theoretical basis for early screening of colon cancer, gene targeting therapy and prognosis.Methods:We validated the expression of miR-139-5p in95paired Colon Cancer and matched adjacent normal tissues and all seven tested human colon cancer cell lines using quantitative PCR. The colon cancer cell lines were transiently transfected with pre-miR-139-5p and pre-miR-Ctrl precursor molecules, used RT-PCR (Real-time Polymerase Chain Reaction) to detect the transfection efficiency; With MTT method to draw the cell growth curve, and through Transwell method to detect the ability of cell invasion and migration, with Flow cytometry instrument to analyze apoptosis and cell cycle of colon cancer cell lines for transfection. Finally, through the Western Blot and PCR assays to verify the related genes expression about cell invasion, migration and apoptosis.Moreover, to discuss the influence of colon cancer growth which transfected with miR-139-5p.Using T-T test method to detect and analyze on the growth of colon cancer cells were transfected miR139-5p and miR-Ctrl, meanwhile including cell proliferation, invasion, metastasis, apoptosis and cell cycle.Results:1. According to the data of RT-PCR analyzed, Our results showed that miR-139-5p was significantly down-regulated in tumor tissues compared with adjacent normal tissues (P<0.0001). The downregulation of miR-139-5p in primary CRCs was significantly associated with tumor distant metastasis (P<0.05). The silence or downregulation of miR-139-5p was also observed in seven tested human colon cancer cell lines compared to normal colon cells, suggesting that miR-139-5p might be a potential tumor suppressor in Colon Cancer.2. For the biological function of colon cancer cells, ectopic expression of miR-139-5p in colon cancer cell lines (DLD1, HCT116, SW1116) significantly suppressed colon cancer cell growth as evidenced by cell viability assay (P<0.0001, P=0.0002, P=0.0022) and colony formation assay (P<0.05in both cell lines).3. Moreover, restoration of miR-139-5p in DLD1and SW1116inhibited cell invasion properties (P=0.0006, P=0.013) and migration (P<0.0001in both cell lines). which were mediated by inhibiting the expression of VIM (SW1116, P=0.0088), MMP1(DLD1P=0.0088), MMP7(DLD1P=0.0035, SW1116P=0.0031) and MMP9.4. miR-139-5p induced DLD1and HCT116apoptosis (P<0.05), concomitantly with up-regulation of key apoptosis genes including cleaved caspase-8and PARP. miR-139-5p also caused cell cycle arrest in G0/G1phase, which was confirmed by the upregulation of key G0/G1phase regulators p21and p27in cell lines (DLD1P=0.0082,P=0.0036, HCT116P=0.025).Conclusion:1. The expression of miR-139-5p was significantly down-regulated in colon cancer tissues and cell lines.2. miR-139-5p played a role as similar as tumor suppressor in colon cancer by inhibiting cell proliferation and promoting apoptosis. It could block cell cycle by regulating the level of P21and P27, which were the important regulatory factors of cell cycle. It induced cell apoptosis through Caspase pathway and miR-139-5p affected the cell migration and invasion by controlling the expression of matrix metalloproteinases.3. miR-139-5p is a novel MicroRNA, it plays a pivotal role in colon cancer development and tumorigenesis through inhibiting cell proliferation, cell migration and invasion, cell cycle and promoting apoptosis. Therefore, miR-139-5p may play a tumor suppressor role in tumorigenesis and development of Colon Cancer.