Smac调控Caspase-3对耐药肺腺癌细胞株生物活性及相关信号的研究

Smac Regulation of Caspase-3 Bioactivity and Related Signal in Drug-resistant Lung Adenocarcinoma Cell Lines

目的:探讨Smac基因调控Caspase-3表达对紫杉醇耐药肺腺癌细胞株生物活性及经典凋亡信号通路的作用机制。方法:取构建好的耐药A549细胞,将其分为A549细胞(LC)组、A549细胞+Smac-NC(SN)组、A549细胞+Smac抑制剂(SI)组、A549细胞+Smac激动剂(SM)组、A549细胞+Caspase-3-NC(CN)组、A549细胞+Caspase-3抑制剂(CI)组、A549细胞+Caspase-3激动剂(CM)组、A549细胞+Smac激动剂+Caspase-3激动剂(MM)组;Real-time PCR法检测正常肺上皮细胞及4种肺腺癌细胞系中Smac、Caspase-3表达水平,将阴性对照、Smac、Caspase-3类似物转染至紫杉醇耐药肺腺癌细胞株,MTT法检测细胞增殖,流式细胞仪检测细胞凋亡,免疫印迹法检测经典凋亡信号通路表达,并分析Smac与Caspase-3的相关性。结果:肺腺癌细胞系中的Smac、Caspase-3 m RNA表达量显著低于正常肺上皮细胞系BEAS-2B (P<0.05),其中A549的Smac、Caspase-3 m RNA值最小(P<0.05),因此选取其作为此次实验细胞;LC组与SN组相比,细胞增殖率、凋亡率及Caspase-3、Bcl-2、Bax、Cyto-C蛋白表达基本无差异(P>0.05),与SN组相比,SI组细胞凋亡率及Caspase-3、Bax、Cyto-C蛋白表达明显降低(P<0.05),增殖率、Bcl-2表达明显升高(P<0.05),与SI组相比,SM组细胞凋亡率及Caspase-3、Bax、Cyto-C蛋白表达明显升高(P<0.05),增殖率、Bcl-2表达明显降低(P<0.05);LC组与CN组相比,细胞增殖率、凋亡率及Caspase-3、Bcl-2、Bax、Cyto-C蛋白表达基本无差异(P>0.05),与CN组相比,CI组细胞凋亡率及Caspase-3、Bax、Cyto-C蛋白表达明显降低(P<0.05),增殖率、Bcl-2表达明显升高(P<0.05),与CI组相比,CM组细胞凋亡率及Caspase-3、Bax、Cyto-C蛋白表达明显升高(P<0.05),增殖率、Bcl-2表达明显降低(P<0.05);SM组与CM组相比,细胞增殖率、凋亡率及Caspase-3、Bcl-2、Bax、Cyto-C蛋白表达基本无差异(P>0.05),与CM组相比,MM组细胞凋亡率及Caspase-3、Bax、Cyto-C蛋白表达明显升高(P<0.05),增殖率、Bcl-2表达明显降低(P<0.05);Smac与Caspase-3呈现正相关(r=0.470,P=0.002),组间具有显著差异。结论:Smac基因可显著改善紫杉醇耐药肺腺癌细胞株细胞生物活性,并激活经典凋亡信号通路,其作用机制可能与调控Caspase-3表达有关。

Objective: To investigate the mechanism of caspase-3 expression regulated by Smac gene on the biological activity and classical apoptotic signaling pathway of paclitaxel-resistant lung adenocarcinoma cells. Methods: The constructed drug-resistant A549 cells were taken, They were divided into A549 cells(LC) group, A549 cells + SMAC-NC(SN) group, A549 cells +Smac inhibitor(SI) group, A549 cells +Smac agonist(SM) group, A549 cells + caspase-3-NC(CN) group, A549 cells +Caspase-3 inhibitor(CI) group,A549 cells +Caspase-3 agonist(CM) group, A549 cells +Smac agonist +Caspase-3 agonist(MM) group;Real-time PCR was used to detect the expression levels of Smac and Caspase-3 in normal lung epithelial cells and 4 kinds of lung adenocarcinoma cell lines. Negative control, Smac and Caspase-3 analogs were transfected into paclitaxel-resistant lung adenocarcinoma cell lines. Cell proliferation was detected by MTT assay and cell apoptosis was detected by flow cytometry. Western blotting was used to detect the expression of classical apoptosis signaling pathway, and the correlation between Smac and Caspase-3 was analyzed. Results: The m RNA expression levels of Smac and caspase-3 in lung adenocarcinoma cell line were significantly lower than those in normal lung epithelial cell line BEAS-2B(P<0.05), and THE m RNA expression levels of Smac and caspase-3 in A549 cell line were the lowest(P<0.05), so it was selected as the experimental cell. Compared with SN group, LC group showed no significant difference in cell proliferation rate, apoptosis rate and protein expression of Caspase-3, Bcl-2, Bax and Cyto-C(P>0.05), while SI group showed significantly lower apoptosis rate and protein expression of Caspase-3, Bax and Cyto-C(P<0.05). The proliferation rate and the expression of Bcl-2 were significantly increased(P<0.05), the apoptosis rate and the expression of Caspase-3, Bax and Cyto-C were significantly increased(P<0.05), and the proliferation rate and the expression of Bcl-2 were significantly decreased(P<0.05) in SM group compared with SI group. Compared with CN group,LC group showed no significant difference in cell proliferation rate, apoptosis rate and protein expression of Caspase-3, Bcl-2, Bax and Cyto-C(P>0.05), CI group showed significantly lower apoptosis rate and protein expression of Caspase-3, Bax and Cyto-C(P<0.05). The proliferation rate and the expression of Bcl-2 were significantly increased(P<0.05). Compared with CI group, the apoptosis rate and the expression of Caspase-3, Bax and Cyto-C in CM group were significantly increased(P<0.05), while the proliferation rate and the expression of Bcl-2 were significantly decreased(P<0.05). Compared with CM group, SM group had no difference in cell proliferation rate, apoptosis rate and protein expression of Caspase-3, Bcl-2, Bax and Cyto-C(P>0.05), while MM group had significantly higher apoptosis rate and protein expression of Caspase-3, Bax and Cyto-C(P<0.05). The proliferation rate and bcl-2 expression were significantly decreased(P<0.05). Smac was positively correlated with Caspase-3(r=0.470, P=0.002), with significant difference between groups.Conclusion: Smac gene can significantly improve the biological activity of paclitaxel-resistant lung adenocarcinoma cells and activate the classical apoptotic signaling pathway, which may be related to the regulation of caspase-3 expression.