Identification And Functional Analysis Of MiR-622Eukaryotic Expression Vector Targeting ING1Gene In Luman Gastric Cancer MKN-45

Background:Gastric cancer is one of the most common malignancies, the mortality rate among the malignant tumor in second place, and a serious threat to people’s life and health. Its origins and development is very complex, involving a variety of immune and molecular mechanism. The molecular mechanism of gastric cancer found that oncogenes and tumor suppressor genes play an important role in development of the gastric cancer, but its expression regulation mechanism still is not clear. MicroRNAs (microRNAs, miRNAs) are new discoveries in recent years a class of small molecule non-coding RNAs, studies have found that they not only play a certain role of regulation in the growth of the organism, development, aging and death, and other biological process, but also play an important role in the occurrence of cancer development. MiR-622is a kind of microRNA of the gastric cancer, MiR-622study found that miR-622directly targeted the cell cycle ING1related gene.AIM:At present, we still lack a comprehensive and in-depth understanding of the pathogenesis of gastric cancer。The discovery of miRNA makes people develop a new understanding of cancerous tumors development。This study aims to construct miR-622eukaryotic expression vector, establish high expression miR-622stably transfected gastric carcinoma cell line, verify the interference effect and function of the ING1gene transfected human gastric cancer cell line MKN-45cells.METHODS: 1.Human gastric cancer cell line MKN-45genome as a template, get the target fragment of the pre-miR-622by PCR。A recombinant plasmid carrying miR-622(pSuper/miR-622) was constructed by insert pre-miR-622target fragment into PSuper. GFP/neo carrier。2. A recombinant plasmid carrying miR-622(pSuper/miR-622) was transfected into MKN-45cells. Cells stably expressing miR-622were selected by using G418. We used MKN-45cells untransfected and those transfected with empty pSuper plasmid as controls. The expression levels of miR-622were detected by real-time PCR in Stably transfected-MKN-45cells。3. Western blot was used to detect of the expression of ING1gene, the CCK-8cell proliferation experiment was used to detect cell growth and cycle experiment was used to verify the influence of highly expressed of in the cell cycle.RESULTS:1. The expression of ING1protein in in empty vector group was10.62±1.04and the expression in pSuper/miR-622group was2.32±0.31.Compared with untransfected MKN-45cells, the expression of ING1protein was an average4.58-fold decrease.2.We evaluated cell growth activity.The result of study was that compared to control group, Over-expression of miR-622leads to cell growth and cell cycle progress.3.We evaluated cell cycle assay.The rsult was that GO/G1phase was21.45±0.16in pSuper/miR-622group and was48.21±0.34in empty vector group in gastric cancer cells; G2/M phase was53.67±0.41in pSuper/miR-622group and was20.27±0.18in empty vector group; compared to pSuper empty vector cells, up-expression of miR-622promote the progress of cell cycle.CONCLUSION:1. The experimental results show that ING1protein is one of miR-622target.2.MiR-622eukaryotic expression vector and stably expressing miR-622MKN-45cells were successfully constructed. It may lay the foundation for the depth study of its basis functions of miR-622in human gastric cancer.