下调TPX2表达对卵巢癌细胞增殖、凋亡的影响及其机制研究

The effect of down-regulation of TPX2 on the proliferation and apoptosis of ovarian cancer cells and its mechanism

目的探讨下调Xklp2靶蛋白(TPX2)表达对卵巢癌细胞增殖、凋亡的影响及其机制。方法以卵巢癌细胞SKO-V3转染TPX2 siRNA和siRNA control分别作为干扰组和阴性组,以不作转染的细胞作为对照组。采用实时荧光定量聚合酶链式反应(qRT-PCR)技术和蛋白质印迹法(Western blot)检测细胞中TPX2 mRNA和TPX2蛋白的表达水平,MTT法检测细胞的增殖情况,平板克隆实验检测细胞克隆形成能力,流式细胞术检测细胞的凋亡情况,Western blot检测细胞中B细胞淋巴瘤/白血病-2(Bcl-2)、活化的含半胱氨酸的天冬氨酸蛋白水解酶3(Cleaved Caspase-3)、增殖细胞核抗原(PCNA)、信号转导与转录因子3(STAT3)、磷酸化的STAT3(p-STAT3)蛋白表达水平。结果阴性组与对照组细胞中TPX2 mRNA和TPX2蛋白的表达水平比较,差异均无统计学意义(P>0.05)。干扰组细胞中TPX2 mRNA和TPX2蛋白的表达水平、光密度(OD)值、克隆形成率均明显低于对照组(P<0.01)。干扰组细胞中Cleaved Caspase-3的水平明显高于对照组,而Bcl-2、PCNA、p-STAT3/STAT3水平均明显低于对照组(P<0.01)。阴性组与对照组细胞的OD值、克隆形成率、凋亡率和Bcl-2、Cleaved Caspase-3、PCNA、STAT3/p-STAT3蛋白水平比较,差异均无统计学意义(P>0.05)。结论下调TPX2表达能够抑制卵巢癌细胞的增殖,诱导卵巢癌细胞的凋亡,降低细胞的克隆形成能力,下调细胞中Bcl-2、PCNA、p-STAT3/STAT3的表达水平,促进细胞中Caspase-3的活化。

Objective To investigate the effect and mechanism of down-regulation the expression of targeting protein for Xenopus kinesin-like protein 2（TPX2） on the proliferation and apoptosis of ovarian cancer cells. Method TPX2 siRNA and siRNA control were transfected into ovarian cancer cells SKO-V3 as interference group and negative group, the non-transfected cells were used as the control group. The expression levels of TPX2 mRNA and TPX2 protein in cells were detected by PCR and Western blot, MTT assay was used to detect cell proliferation, the colony forming ability of cells was determined by plate cloning test, cell apoptosis was analyzed by flow cytometry, Western blot was utilized to detect the expression level of Bcl-2, Cleaved Caspase-3, PCNA, STAT3 and p-STAT3 in cells. Result The expression levels of TPX2 mRNA and TPX2 protein in the negative group were similar with those in control group, the difference was not statistically significant（P〉0.05）. In the cells of interference group, the expression level of TPX2 protein and mRNA,optical density（OD） value, colony formation rate were significantly lower than those in control group（P〈0.01）. The level of Cleaved Caspase-3 in interference group was significantly higher than that in control group, while the levels of Bcl-2, PCNA and p-STAT3/STAT3 were significantly lower compared with control group（P〈0.01）. The OD value, colony formation rate, apoptosis rate and Bcl-2, Cleaved Caspase-3, PCNA, STAT3/p-STAT3 protein levels in the negative group were comparable with those in control group, with no significant difference observed（P〉0.05）. Conclusion Down-regulation the expression of TPX2 could inhibit the proliferation of ovarian cancer cells, inducing apoptosis, reducing colony formation ability, down regulating the level of Bcl-2, PCNA and p-STAT3/STAT3, and promoting Caspase-3 activation in cells.