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将合成的miR-34a成熟序列转染p53–/–的非小细胞肺癌H1299细胞,探究外源性的miR-34a是否影响p53缺失细胞的生长、凋亡和衰老以及其作用机制。MTT检测细胞的生长与存活,藻红B染色检测细胞的死亡,Annexin V/PI染色检测细胞凋亡,β-半乳糖苷酶染色试剂盒检测细胞的衰老,Western blot检测与细胞凋亡和周期相关的Bcl-2、Puma、Cdk4和E2F3蛋白的表达。结果显示,miR-34a转染组和阴性对照组相比,存活率明显降低,且48 h较24 h更显著,24 h和48 h存活率分别为79.94%、64.83%;细胞死亡和凋亡的分析结果表明,外源性miR-34a可以促进细胞死亡和凋亡;此外,细胞还出现了明显的衰老,并且检测到凋亡、衰老相关的Bcl-2、Puma、E2F3和Cdk4蛋白的表达下调。miR-34a可以下调p53下游相应靶蛋白的表达,通过部分补救p53通路或p53非依赖途径促进细胞的凋亡、衰老并抑制细胞的增殖。
The synthetic miR-34a mature sequence was transfected into p53 - / - non-small cell lung cancer H1299 cells to explore whether extrinsic miR-34a could affect the growth, apoptosis and aging of p53-deficient cells and its mechanism. The cell growth and survival were detected by MTT assay. The cell death was detected by algal B staining. Cell apoptosis was detected by Annexin V / PI staining. Cell cycle was detected by Western blot. Related Bcl-2, Puma, Cdk4 and E2F3 protein expression. The results showed that compared with the negative control group, the survival rate of miR-34a transfection group was significantly lower than that of the negative control group, and 48 h was more significant than 24 h, the survival rates at 24 h and 48 h were 79.94%, 64.83% respectively; cell death and apoptosis The results showed that exogenous miR-34a could promote cell death and apoptosis. In addition, the cells also showed obvious aging, and the expression of apoptosis, aging-related Bcl-2, Puma, E2F3 and Cdk4 protein were detected Down. miR-34a down-regulates the expression of the corresponding target protein downstream of p53 and promotes apoptosis, senescence and cell proliferation through partial salvage of p53 pathway or p53-independent pathway.