谷氧还蛋白3基因表达对肺癌细胞增殖和凋亡的影响及其机制

Proliferation and apoptosis of lung cancer cells regulated by gultaredoxin 3

目的观察抑制谷氧还蛋白3（GLRX3）基因表达对肺癌细胞增殖和凋亡的影响，并探讨其机制。方法Western blot检测人胚肺成纤维细胞MRC5及肺癌A427、A549、PC9、H1299细胞中GLRX3的蛋白表达。采用RNA干扰技术，将合成的GLRX3 siRNA转染A549细胞（实验组），转染阴性siRNA的A549细胞作为阴性组，不经特殊处理的A549细胞为空白组。Western blot检测转染48 h各组细胞中GLRX3、活化的含半胱氨酸的天冬氨酸蛋白水解酶3（cleaved caspase-3）、信号转导与转录因子3（STAT3）和p-STAT3的蛋白表达，细胞计数试剂盒8（CCK-8）法检测转染24、48和72 h细胞的增殖情况，流式细胞术检测转染48 h细胞的凋亡情况。结果人胚肺成纤维细胞MRC5及肺癌A427、A549、PC9、H1299细胞中GLRX3的蛋白表达量分别为0.094±0.010、0.282±0.021、0.551±0.045、0.423±0.039和0.454±0.036，肺癌A427、A549、PC9、H1299细胞中GLRX3的蛋白表达量均显著高于MRC5细胞（均P〈0.01）。转染A549细胞24、48和72 h，实验组与空白组吸光度差异均有统计学意义（均P〈0.01）。转染A549细胞48 h，空白组、阴性组和实验组细胞中GLRX3的蛋白表达量分别为0.311±0.029、0.328±0.032和0.103±0.012，实验组显著低于空白组（P〈0.01），阴性组与空白组差异无统计学意义（P〉0.05）；空白组、阴性组和实验组细胞凋亡率分别为（1.65±0.22）%、（1.42±0.26）%和（9.52±0.56）%，实验组细胞凋亡率显著高于空白组（P〈0.01）；实验组cleaved caspase-3的蛋白表达显著高于空白组（P〈0.01），p-STAT3的蛋白表达显著低于空白组（P〈0.01），而STAT3蛋白表达各组间差异无统计学意义（P〉0.05）。结论抑制GLRX3基因表达，可通过上调cleaved caspase-3的表达、下调STAT3信号通路，抑制肺癌细胞增殖，诱导细胞凋亡。

ObjectiveTo investigate the effects of inhibiting glutaredoxin 3（GLRX3） expression on proliferation and apoptosis of lung cancer cells.MethodsWestern blotting was used to detect the expressions of GLRX3 protein in human embryonic lung fibroblast MRC5 and lung cancer cells, including A427, A549, PC9 and H1299. GLRX3-targeted siRNA （experimental group） and negative siRNA （negative group） were transfected into A549 cells, and the cells without special treatment were blank group. The protein expression levels of GLRX3, cleaved cysteinyl aspartate specific proteinase 3（cleaved caspase-3）, signal transducers and activators of transcription 3（STAT3）, phosphorylated STAT3（p-STAT3） in each group at 48 hours after transfection were measured by Western blotting. The proliferation ability of differently treated cells at 24 hours, 48 hours and 72 hours after transfection were detected by CCK-8 array. The cell apoptosis at 48 hours after transfection was evaluated by flow cytometry.ResultsThe protein expression levels of GLRX3 in MRC5, A427, A549, PC9 and H1299 were 0.094±0.010, 0.282±0.021, 0.551±0.045, 0.423±0.039 and 0.454±0.036, respectively. The protein expressions of GLRX3 in tested lung cancer cells were significantly higher than that of MRC5 cells （all P〈0.01）. The GLRX3 protein expressions in blank group, negative control group and experimental group at 48 hours after transfection were 0.311±0.029, 0.328±0.032 and 0.103±0.012, respectively. GLRX3 protein expression level of experimental group in A549 cells was significantly lower than that of control group （P〈0.01）, whilewithout statistical difference between the negative group and blank group （P〉0.05）. A values of experimental group at 24, 48 and 72 hours after transfection in A549 cells were significantly different from those of blank group （all P〈0.01）. Percent of apoptotic cells in the experimental group was （9.52±0.56）%, which was significantly higher than that of blank group [（1.65±0.22）%] and negative control group [（1.42±0.26）%, all P〈0.01]. Consistently, compared with blank group, the cleaved caspase-3 markedly increased in the experimental group （P〈0.01）. The protein expression of p-STAT3 in the experimental group was significantly lower than the blank group （P〈0.01）, while no significant difference of STAT3 protein expression was observed among all the groups （P〉0.05）.Conclusions Inhibition of GLRX3 gene expression can inhibit the proliferation of lung cancer cells and induce cell apoptosis through up-regulating cleaved caspase-3 expression and down-regulating STAT3 signaling pathway.