The Molecular Mechanisim Of MicroRNA-590-5p As Potential Oncogenic MicroRNA Of Human Colon Cancer Cells By Targeting PTEN

Part one:Expression analysis and biological function study of miR-590-5p in colon cancerObjective Analysis of the expression level of miR-590-5p in colon and colon cancer cell lines.Study on the effect of inhibiting the expression of miR-590-5p on the growth of colon cancer cells.Method 20 cases of colon cancer and corresponding paracancerous tissue were collected,and PCR technique was used to detect the expression level of miR-590-5p in colon cancer clinical samples.The normal colonic epithelial cell line(NCM460)and a variety of colon cancer cell lines(HCT116,SW620 and HT29)were cultured,and PCR technique was used to detect the expression level of miR-590-5p in cell lines.MiR-590-5p inhibitor and corresponding negative control sequence(NC)were transfected into colon cancer cell lines HCT116 and SW620,then the expression of miR-590-5p in colon cancer cell line after transfection was detected by PCR,the proliferation of colon cancer cell lines after transfection was detected by CCK-8,flow cytometry was used to detect the level of cell apoptosis and the distribution of cell cycle.Result The expression of miR-590-5p in colon cancer was significantly higher than that in paracancerous tissue(P<0,01).The expression level of miR-590-5p in HCT116,SW620 and HT29 is about 3-5 times as much as NCM460(P<0.01).In SW620 and HCT116,transfection of miR-590-5p inhibitor could significantly inhibit the expression of miR-590-5p(P<0.01).CCK-8 and flow cytometry showed that miR-590-5p inhibitor could significantly inhibit SW620 and HCT116 cell activity(P<0.01),induce apoptosis and arrest cell cycle in G0/G1 phase.Conclusion MiR-590-5p is highly expressed in both colon and colon cancercell lines.Inhibition of miR-590-5p expression can inhibit the growth of colon cancer cells.MiR-590-5p inhibitor inhibits the growth of colon cancer cells by inducing cellapoptosis and cell cycle arrest.Part two:Identification of miR-590-5p target gene and its proliferative function and molecular mechanisms n colon cancer cellsObjective Analysis and verification that PTEN is the target gene for the action of miR-590-5p in colon cancer cells.Detection of miR-590-5p on the regulation of PI3K/AKT/mTOR signaling pathway.Verification that the role and molecular mechanism of microRNA-590-5p in tumorigenesis of colon cancer cells in nude mice.Method The candidate target gene PTEN was screened by bioinformatics analysis of the binding site of the miR-590-5p target gene.Double luciferase reporter gene was used to detect luciferase activity in PTEN wild type and mutant plasmids which were transfected by miR-590-5p inhibitor and miR-590-5p mimic respectively.The expression of PTEN in colon cancer was detected by PCR method.HCT116 and SW620 cells were transfected with miR-590-5p inhibitor,miR-590-5p inhibitor+si-PTEN respectively,then cell proliferation and apoptosis were detected by CCK8 and flow cytometry.HCT116 and SW620 cells were transfected with miR-590-5p mimic and miR-590-5p inhibitor respectively,then western blot technique was used to detect the expression of PTEN and signaling pathway related proteins(AKT,p-AKT,mTOR,p-mTOR).36 nude mice were divided into 3 groups randomly.HCT116,HCT116 + miR-590-5p milic,HCT116＋miR-590-5p inhibitor(5×106)were subcutaneously inoculated in each group respectively.Two weeks later,six nude mice in each group were intraperitoneally injected with oxaliplatin(5mg/kg).After 4 weeks,nude mice were executed and the volume and weight of tumors were recorded.The Expression of-PTEN in tumor tissues was detect by immunohistochemistry,the expression of microRNA-590-5p,PTEN and AKT in tumor tissues was detect by PCR.Western blot was used to detect the expression of PTEN,AKT,p-AKT?mTOR and p-mTOR in tumor tissues.Result The PTEN gene exists the potential binding site of miR-590-5p.Dual luciferase reporter assay showed that in the wild type PTEN plasmid miR-590-5p mimic could significantly inhibit the luciferase activity,miR-590-5p inhibitor can significantly increase the luciferase activity(P<0.01),but PTEN mutant plasmid had no effect(P>0.05).PCR detection showed that PTEN was expressed at a low level in colon cancer.CCK-8 and flow cytometry showed that miR-590-5p inhibitor significantly inhibited the proliferation of colon cancer cells and induced apoptosis(P<0.01),and the effect of si-PTEN was compensated after transfection.In HCT116 and SW620,miR-590-5p mimic significantly inhibited the expression of PTEN and promoted the expression of p-AKT,p-mTOR protein,the effect of microRNA-590-5p inhibitor was opposite(P<0.01).Nude mice experiments showed that microRNA-590-5p mimic promoted the tumorigenesis of HCT116.The expression of PTEN in tumor tissue was down-regulated,while the expression of p-AKT and p-mTOR in tumor tissue were up-regulated.The effect of microRNA-590-5p inhibitor was opposite.Conclusion In colon cancer,PTEN is the target gene for miR-590-5p.MiR-590-5p may promote the function of the oncogenic gene by inhibiting the targeting of PTEN and then promoting the PI3K/AKT/mTOR signaling pathway.