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目的研究microRNA-424-5p(miR-424-5p)对人胃癌细胞SGC-7901自噬的影响及可能机制。方法培养人正常胃黏膜上皮细胞GES和人胃癌细胞SGC-7901,应用实时定量PCR方法检测miR-424-5p的表达水平;应用Lipofectamine LTX试剂将化学合成的miR-424-5p agomir和miR-424-5p antagomir转染人胃癌细胞SGC-7901,验证转染效率后应用MTT方法检测细胞活力;应用免疫荧光法检测自噬标记物微管相关蛋白轻链-3(LC3)和自噬降解底物p62/SQSTM1在SGC-7901细胞的分布;应用Western blot方法检测LC3-Ⅱ、p62/SQSTM1和自噬相关蛋白Beclin1的蛋白表达水平。结果 miR-424-5p在人胃癌细胞SGC-7901中呈低表达,miR-424-5p过表达显著抑制人胃癌细胞SGC-7901的细胞活力,miR-424-5p表达沉默的作用效果与之相反。miR-424-5p过表达后自噬标记物LC3在SGC-7901细胞的胞浆内呈点状分布,荧光表达显著增强,并且LC3-II的蛋白表达水平显著上调,同时伴有自噬降解底物p62/SQSTM1在细胞浆内荧光表达显著减弱,蛋白表达下调,此外,自噬相关蛋白Beclin1的蛋白表达水平显著上调;miR-424-5p表达沉默的作用效果与之相反。结论 miR-424-5p过表达明显抑制人胃癌细胞SGC-7901的细胞活力,诱导细胞发生自噬,其机制之一可能与自噬相关蛋白Beclin1上调有关。
Objective To study the effect of microRNA-424-5p (miR-424-5p) on autophagy in human gastric cancer cell line SGC-7901 and its possible mechanism. Methods Human gastric epithelial cells (GES) and human gastric cancer cell line SGC-7901 were cultured and the expression of miR-424-5p was detected by real-time quantitative PCR. Chemically synthesized miR-424-5p agomir and miR-424 -5p antagomir was transfected into human gastric cancer cell line SGC-7901, and the cell viability was detected by MTT assay after transfection efficiency was verified. The autophagy marker microtubule-associated protein light chain-3 (LC3) and autophagy degradation substrate p62 / SQSTM1 in SGC-7901 cells. Western blot was used to detect the expression of LC3-Ⅱ, p62 / SQSTM1 and autophagy-related protein Beclin1. Results miR-424-5p was lowly expressed in human gastric cancer cell line SGC-7901 and over-expression of miR-424-5p significantly inhibited the cell viability of SGC-7901 human gastric cancer cell line SGC-7901 . The expression of LC3-II was significantly upregulated in the cytoplasm of SGC-7901 cells after LC3-II expression of miR-424-5p was over-expressed. The expression of p62 / SQSTM1 in the cytoplasm decreased significantly, and the protein expression was down-regulated. In addition, Beclin1 protein expression was significantly up-regulated. The effect of miR-424-5p silencing was opposite. Conclusion Overexpression of miR-424-5p can significantly inhibit the cell viability of SGC-7901 cells and induce autophagy. One of the mechanisms may be related to the up-regulation of autophagy-related protein Beclin1.