过表达白血病抑制因子增强子宫内膜癌细胞对化疗药物的耐受

Overexpression of leukemia inhibitory factor enhances chemotherapy tolerance of endometrial cancer cells in vitro

目的探讨在体外环境中,子宫内膜癌细胞高表达白血病抑制因子(LIF)后,对顺铂及紫杉醇的敏感性变化。方法利用慢病毒在子宫内膜癌细胞(HEC-1B和RL95-2)中建立稳定过表达LIF的细胞模型,以空载体慢病毒感染细胞作为对照组,用逆转录荧光定量聚合酶链反应(RT-qPCR)和酶联免疫吸附试验验证LIF的表达。利用CCK-8观察LIF过表达对癌细胞活性的影响,利用Annexin V-FITC/PI法和JC-1法观察LIF过表达后,顺铂及紫杉醇诱导癌细胞凋亡及线粒体膜电位下降的情况。利用免疫印迹观察LIF过表达后,癌细胞中Bcl-2家族及STAT3通路蛋白的表达。利用荧光素酶报告基因活性检测观察LIF过表达后,癌细胞STAT3转录活性的变化。在过表达LIF的两子宫内膜癌细胞中沉默STAT3,以无义小干扰RNA序列转染细胞为对照组,观察顺铂及紫杉醇对细胞的杀伤作用。结果过表达组的子宫内膜癌细胞(HEC-1B和RL95-2)较对照组表达更高水平的HLF mRNA及分泌更多的HLF蛋白(P<0.05)。LIF的过表达不影响两子宫内膜癌细胞的体外增殖活性(P>0.05),但能增加癌细胞在顺铂及紫杉醇作用下的存活率和线粒体膜电位(P<0.05)。过表达LIF能增加两子宫内膜癌细胞中Bcl-2、Bcl-xL和p-STAT3的水平,降低Bax和Bad的水平,而不影响STAT3的水平;同时STAT3的转录活性增强(P<0.05)。在稳定表达LIF的两子宫内膜癌细胞中,沉默STAT3能显著增加顺铂及紫杉醇对细胞的杀伤作用(P<0.05)。结论在体外环境中,稳定高表达LIF能增强宫内膜癌细胞对顺铂及紫杉醇的抵抗。

Objective To investigate the effect of overexpression of leukemia inhibitory factor(LIF) on cisplatin and paclitaxel resistance of endometrial cancer cells in vitro. Methods Endometrial cancer cell lines HEC-1 B and RL95-2 were infected with a recombinant lentivirus to overexpress LIF, and the changes in LIF expression was verified using RT-qPCR and ELISA. The viability of the LIF-overexpressing cells was assessed using CCK-8 assay, and the cell apoptosis and changes in mitochondrial membrane potential in response to cisplatin or paclitaxel treatment were analyzed with annexin V-FITC/PI staining and JC-1 assay, respectively. The effect of LIF overexpression on the expressions of Bcl-2 family proteins and STAT3 pathway was evaluated using Western blotting;dual-luciferase reporter gene assay was employed to detect the transcriptional activity of STAT3. The effect of STAT3 silencing on apoptosis of the LIF-overexpressing cells induced by cisplatin or paclitaxel was investigated. Results The cell lines infected with the recombinant lentivirus showed significantly increased mRNA and protein levels of LIF(P<0.05) without obvious changes in the cell viability(P>0.05). LIF overexpression significantly attenuated cisplatin-or paclitaxel-induced apoptosis of the endometrial cancer cells(P<0.05) and markedly increased mitochondrial membrane potential of the cells(P<0.05). The expressions of Bcl-2, Bcl-xL and p-STAT3 proteins increased obviously while the expressions of Bax, Bad and STAT3 either decreased or showed no obvious changes in the LIF-overexpressing cells.Overexpressing LIF significantly enhanced the transcriptional activity of STAT3(P<0.05), and silencing STAT3 obviously enhanced apoptosis of the endometrial cancer cells overexpressing LIF(P<0.05). Conclusions Overexpression of LIF can enhance cisplatin and paclitaxel resistance to endometrial cancer cells in vitro.