Study On The Influence And Mechanism Of MiR-200a And MiR-141on Biologocal Characters Of Human Gastric Cancer Cell Line SGC7901

Objectives:To explore the effects of transfection of miR-200a mimics and miR-141mimics on proliferation, invasion and migration of human gastric cancer cell line SGC7901biological behavior changes,and to investigate its possible mechanism.Methods:Oligonucleotides miR-200a mimics and miR-141mimics mediated by lipofectamine were transfected to human gastric cancer cell line SGC7901by targeting alone or combinated; without transfection (Control), performing transfection with nonsense sequence (Scrambled) were established. Real time PCR was conducted to detect the expressions of miRNA in these groups after the transfection. MTT method was used to evalute the cell proliferation rate; the invasion and migration capability of cells were evaluated by using Transwell chamber model and wound healing assay, respectively; while the expression of E-cadherin, ZEB2, N-cadherin, MMP-9were determined by Western blot analysis and Immunofluorescence assay.Results:1. Forty-eight hours after the transfection, Real time PCR showed that the relative expression of miRNA in the miR-200a Mimi、miR-141Mimi and Combined groups were upregulated compared to Control group and Scrambled group, and the expression of miRNA in the Combined groups was more than miR-200a Mimi、 miR-141Mimi groups.The results confirmed that the expressions of miR-200a and miR-141were successfully regulated in human gastric cancer cell line SGC7901after the transfection and meet the requirement of the follow experiments.2. MTT assay indicated the cell proliferation rate two days after the transfection in the miR-200a Mimi、miR-141Mimi and Combined groups were significantly reduced, as compared with those in Control group and Scrambled group, the diference was statistical significance (P<0.05), in addition, the cell proliferation rate two days after the transfection in the Combined transfected group was significantly lower than in the miR-200a Mimi、miR-141Mimi group, the diference was statistical significance(P<0.05).3. Transwell chamber assay demonstrated the invasive capability of cells through the martrigel coated transwell membrane of miR-200a Mimi group (76.33±3.79), miR-141Mimi group (65.33±4.72), Combined group (29.67±2.08) were significantly lower than Control group(137.67±5.13) and Scrambled group(136.67±6.03), the diference was statistical significance(P<0.05), the invasive capability of Combined group was significantly reduced as compared with those in the miR-200a Mimi、miR-141Mimi group, the diference was statistical significance(P<0.05).4. Tested by wound healing assay, the migration ability of miR-200a Mimi、 miR-141Mimi and Combined groups were more poor than Control group and Scrambled group, the migration ability of Combined group was significantly inhibited as compared with those in the miR-200a Mimi、miR-141Mimi group.5. Western blot analysis detected the protein expression of miR-200a Mimi、 miR-141Mimi and Combined groups,while overexpression miR-200a and miR-141increased E-cadherin protein level and reduced ZEB2, N-cadherin, MMP-9protein levels, the protein expression of Combined group was significant, the diference was statistical significance(P<0.05).6. Immunofluorescence staining and confocal laser scanning microscopy revealed that N-cadherin and MMP-9decreased in cell membranes and nuclear ZEB2also decreased while E-cadherin increased in cell membranes in the miR-200a Mimi、miR-141Mimi and Combined groups as compared in the Control group and Scrambled group, the protein expression of Combined groups was obvious.Conclusions:1. The up-regulated expression of miR-200a and miR-141could significantly inhibit the invasion and migration ability of human gastric cancer cell line SGC7901, the invasion and migration effect was reduced obviously when combinated.2. miR-200could suppress the expression of ZEB2to increase the expression of E-cadherin and decrease the expression of N-cadherin-. MMP-9.ZEB2may promote the invasion and metastasis of gastric cancer cell via the regulation of EMT.The downregulation of ZEB2expression could block the occurrence of EMT.