miR-199a-3p通过上调NET1表达促进胃癌细胞增殖

miR-199a-3p promotes proliferation of gastric cancer cells via up-regulating expression of NET1

目的:探究miR-199a-3p对肿瘤相关基因NET1表达的影响,进一步明确miR-199a-3p、NET1与人胃癌细胞株增殖的关系。方法:采用qRT-PCR检测人胃癌细胞株BCG-823、MKN-28以及正常胃黏膜上皮细胞GES-1中miR-199a-3p的表达情况,CCK-8法检测miR-199a-3p对BCG-823细胞增殖的影响;构建重组荧光素酶报告基因pISO-NET1-3′-UTR,转染后双荧光素酶法检测荧光活性;转染miR-199a-3p和Anti-miR-199a-3p表达质粒后分别采用Westernblot以及qRT-PCR检测BCG-823细胞中NET1的表达水平;NET1干扰质粒与miR-199a-3p质粒共转染后,流式细胞技术、CCK-8法分别检测BCG-823细胞凋亡及增殖情况。结果:人胃癌细胞株BCG-823、MKN-28中miR-199a-3p表达水平要显著高于GES-1细胞,且上调miR-199a-3p表达能够显著促进BCG-823、MKN-28细胞株增殖;双荧光素酶检测结果显示,共转染miR-199a-3p与pISO-Net1-3′-UTR后细胞内荧光活性要显著高于对照组(P<0.01);BCG-823细胞株转染miR-199a-3p质粒后其细胞内NET1蛋白以及mRNA的表达水平均要显著高于空白转染组;此外,转染NET1干扰质粒能够显著抑制BCG-823细胞的增殖并促进细胞凋亡(P<0.01)。结论:miR-199a-3p在胃癌细胞中高表达,且可通过上调NET1表达来促进胃癌细胞增殖。

Objective:To investigate the effect of miR-199a-3p on the expression of tumor-associated gene NET1,and further clarify the relationship between miR-199a-3p,NET1 and the proliferation of human gastric cancer cell lines.Methods:The expression of miR-199a-3p in human gastric cancer cell lines BCG-823,MKN-28 and normal gastric mucosa epithelial cells GES-1 were detected by qRT-PCR,CCK-8 assay was used to detect the effect of miR-199a-3p on the proliferation of BCG-823 cell.The recombinant luciferase reporter gene pISO-NET1-3′-UTR was constructed to detected the fluorescence activity by double luciferase assay after transfection.The expression levels of NET1 were detected by Western blot and qRT-PCR after transfection of miR-199a-3p and Anti-miR-199a-3p.The apoptosis and proliferation of BCG-823 cells were detected by flow cytometry and CCK-8 assay after the co-transfection of the interfering plasmid with miR-199a-3p.Results:The expression level of miR-199a-3p in BCG-823 and MKN-28 cells were significantly higher than GES-1 cells,BCG-823 and MKN-28 cells were significantly proliferation after up-regulation of miR-199a-3p.Double luciferase assay results showed that the intracellular fluorescence activity was significantly higher than control group(P<0.01)after co-transfection of miR-199a-3p and pISO-NET1-3′-UTR.After transfection of mir-199a-3p with BCG-823 cell,the expression levels of NET1 protein and mRNA were significantly higher than blank transfection group.In addition,the interference plasmid transfected with NET1 significantly inhibited the proliferation of BCG-823 cells and promoted apoptosis(P<0.01).Conclusion:miR-199a-3p was highly expressed in gastric cancer cells,and it can promote the proliferation of gastric cancer cells via up-regulating the expression of NET1.