摘要
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 investigate the effect and potential mechanism of microRNA-424-5p （miR-424- 5p） on autophagy of human gastric cancer cells SGC-7901. Methods Human gastric mucosal epithelial cells coES and human gastric cancer cells SGC-7901 were cultured. The expression levels of miR-424-5p were detected by Real-time PCR. Lipofectamine LTX was applied to transfecting gastric cancer cells SGC-7901 with miR-424-5p agomir and miR- 424-5p antagomir. The transfection efficiency was tested by Real-time PCR, MTT was used to detect the viability of gastric cancer cells SGC-7901. The expression and distribution of autophagy marker LC3 and autophagy degradation substrate p62/ SQSTM1 were assessed by fluorescence microscopy. Western blot was used to determine the expression of LC3-Ⅱ , p62/ SQSTM1 and autophagy-related protein Bechlinl. Results miR-424-5p was lowly expressed in human gastric cancer cells SGC-7901. The overexpression of miR-424-5p inhibited the cell viability of SGC-7901 cells, but quite opposite for miR-424-5p silence. The overexpression of miR-424-5p significantly increased LC3-stained autophagic compartments with punctate dots in SGC-7901 ceils, upregulated the expression level of LC3-Ⅱ protein, and attenuated obviously the expression of p62/SQSTM1, increased the expression of Beclinl protein was significant, but decreased for miR-424-5p silence. Conclusion The overexpression of miR-424-5p inhibited the cell viability and induced the autophagy of human gastric cancer ceils SGC-7901, which might be related to upregulation of Beclin 1.
出处
《解剖科学进展》
2017年第1期1-5,共5页
Progress of Anatomical Sciences
基金
国家自然科学基金(81402573)
