MiRNA-493Decreases Cell Proliferation Ability And Migration Ability Lung Cancer Cells By Down-regulating E2F1and RhoC

Background and objectiveLung cancer is one of the most common malignancies. The mortality and morbidity of this disease have increased dramatically during the past decade. According to the biological characteristics, lung cancer is divided into two main categories by the World Health Organization: non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). NSCLC accounts for more than80%of all lung cancer cases. In recent years, with the improvement of treatment, Lung cancer treatment has made considerable progress. But the prognosis of lung cancer is poor,5-year survival rate of less than15%. However, the majority of NSCLC patients diagnosed at late stage and further invasion, metastasis and resistance to chemotherapy during treatment all contribute to the poor treatment efficacy. The mechanism of metastasis of lung cancer has yielded great results, but it’s still not clear. More and more research evidence suggests that miRNAs play an important role in tumor invasion and metastasis. Previously, our research group found that the expression levels of miR-493in lung cancer cells are lower than that of normal lung cells. And it is reported that miR-493can inhibit the metastasis and proliferation of colon cancer and prostate cancer cells, playing the role of tumor suppressor genes. Based on these, our research group has established a cell line:95D lung cancer cell line that stably expresses miRNA-493and investigated the impact of miRNA-493on the biological function of95D lung cancer cells and explored its possible mechanisms.Methods(1)miRNA-493lentivirus vectors and empty vectors as negative control were constructed and transfected into95D cells respectively to establish cell line95D/miR-493stably expressing miR-493and control cell95D/control. And real-time PCR was utilized to detect the expression level of miR-493in these cells.(2) After three cell groups were established:95D cells,95D/control cells transfected with empty lentivirus vectors and95D/miR-493cells expressing miR-493,cell cycles were detected by flat-cloning experiments and flow cytometry assay; the proliferation capacity differences among the three cell groups were investigated by cell growth curve created by MTS; Sensitivity to cisplatin and oxaliplatin of the cells was detected by MTS; the apoptosis of cells in each group were assessed through Hoechst staining; cell invasion and metastasis was investigated by Transwell Migration Assay and cell migration assay.(3) Use RT-PCR and Western blot assays to detect the expression level of RhoC and FZD4and IGF1R in95D cell and95D/control cell and95D/miR-493cells.(4)95D cells were transiently transfected by RhoC-si-RNA. The ability of cell proliferation was tested by MTS. Invasive ability was investigated by Transwell assay. Western blot was used to detect the expression of MMP2and MMP9in95D/miR-493and95D/si-RhoC and their respective control cells.(5) By using bioinformatics software and considering preliminary experimental results of biological functions, we screen out potential miR-493’s target gene E2F1. Real time PCR and Western blot were used to detect the changes of E2F1after transfecting miR-493. A dual-luciferase reportor construct with or without3’UTR of E2F1was transfected into293cells in the presence of a miR-493minic, and Firfly and Renila luciferase activities were measured after24h.(6)95D cells were transiently transfected by E2F1-si-RNA. Cell cycle was detected by flowcytometry. The ability of cell proliferation was tested by MTS. Western blot was used to detect the expression of P21and cyclinD2and ERK and P-ERK in95D/miR-493and95D/si-E2F1and their respective control cells. Results(1)Set the relative expression level of miR-493in95D group as1, the expression level of miR-493in95D/con group and95D/miR-493group is (1.02±0.34) and (10.65±0.59) respectively. Compared to95D group and group95D/con, the relative expression level of miR-493in95D/miR-493group increased significantly, the difference was statistically significant.(2) For the95D/miR-493cells, Colony formation rate significantly reduced and proliferation slowed down, the proportion of S-phase cells decreased while a significant increase in the proportion of cells in G0/G1phase; Cell invasion and metastasis weakened; there is no significant difference in the drug sensitivity; the percentage of apoptotic cells did not show significant data.(3) The expression levels of RhoC was sidnificantly decreased compared to control cells, however miR-493did not repress the expression levels of FZD4and IGF1R. Cell migration was significantly decreased in si-RhoC transfectants compared with control cells, there is no significant difference in the ability of cell proliferation. The expression levels of MMP2and MMP9were significantly decreased in95D/miR-493and95D/si-RhoC compared with control cells.(4) By using bioinformatics software and considering preliminary experimental results of biological functions, we found that E2F1is target oncogenes of miR-493. The expression levels of E2F1was sidnificantly decreased compared to control cells.Dual-Luciferase reporter gene analysis showed that E2F1is a target gene of miR-493.(5)The ability of cell proliferation was significantly decreased in si-E2F1transfectants compared with control cells, and the proportion of S-phase cells decreased while a significant increase in the proportion of cells in G0/G1phase. Western blot results showed that in95D/miR-493and95D/si-E2F1cells, expression level of P21increased while that of cyclinD2, P-ERK reduced.Conclusion(1)Cell line95D/miR-493with stable overexpression of miR-493and cell line95D/control were established for the first time.(2)Mir-493can decrease cell proliferation ability and migration ability in95D human lung cancer cells.(3) Tumor suppressor miR-493decreases cell migration ability in95D lung cancer cells by targeting RhoC through down-regulating MMP2and MMP9.(4) Tumor suppressor miR-493decreases cell proliferation ability in95D lung cancer cells by targeting E2F1through down-regulating cyclinD2and P-ERK and up-regulating P21.(5) Tumor suppressor miR-493may play different roles in different cells and tissues by targeting different genes.