端粒酶逆转录酶线粒体转位促进肝癌细胞干性及耐药的研究

目的 探讨人端粒酶逆转录酶(human telomerase reverse enzyme,hTERT)线粒体转位对肝癌细胞干性及耐药的影响.方法 采用大剂量顺铂(CDDP)冲击、间歇诱导的方法诱导肝癌细胞HepG2、Huh7耐药,构建耐药细胞株HepG2/CDDP、Huh7/CDDP,CCK-8检测细胞耐药能力;Western blot检测细胞线粒体hTERT表达情况;激光共聚焦显微镜观察细胞线粒体膜电位;流式细胞术检测耐药细胞干性相关蛋白CD133、EpCAM表达情况;克隆形成实验检测细胞克隆形成能力;Western blot检测耐药细胞干性相关因子OCT-4蛋白表达水平变化.结果 与亲本细胞HepG2耐药指数(7.69±0.86)μg/mL及Huh7 (7.18±0.35) μg/mL]相比,耐药细胞株对CDDP耐药指数明显增加HepG2 (41.16±0.42) μg/mL,Huh7 (33.48±0.33)μg/mL,P＜0.01],经顺铂(CDDP)诱导耐药细胞线粒体hTERT表达显著升高,线粒体膜电位增强,CD133+细胞比例HepG2 (1.44±0.41),HepG2/CDDP (23.15±1.55),P＜0.01]及EpCAM+细胞比例HepG2 (0.85 ±0.10),HepG2/CDDP (3.96 ±0.10),P＜0.01]增加,克隆形成能力增强,干性相关因子OCT4蛋白表达水平增加.结论 经顺铂诱导的肝癌耐药细胞线粒体hTERT转位显著增加,且具有明显的肿瘤干细胞特征.

Mitochondrial translocation of human telomerase reverse enzyme promotes stemness and drug-resistance of hepatocellular carcinoma cells

Objective To determine the effect of mitochondrial translocation of human telomerase reverse enzyme (hTERT) on the stemness and drug-resistance of hepatocellular carcinoma (HCC) cells.Methods Human HCC cell lines HepG2 and Huh7 were exposed intermittently to cisplatin (CDDP) to obtain cisplatin-resistant cell lines HepG2/CDDP and Huh7/CDDP.CCK-8 assay was employed to detect cell sensitivity to CDDP.Western blotting was adopted to detect hTERT protein level in the mitochondria,and mitochondrial membrane potential was measured by confocal laser scanning microscopy.Flow cytometry was used to detect the expression of cell stemness-related proteins CD133 and EpCAM,and the colony formation ability of the cells was assessed with colony formation assay;Western blotting was used to detect the expression level of stemness-related factor OCT-4.Results The resistance indexes of CDDP-resistant HepG2 and Huh7 cells were significantly increased compared with their parental cells (HepG2:41.16 ± 0.42 vs 7.69 ± 0.86 μg/mL;Huh7:33.48 ± 0.33 vs 7.18 ± 0.35 μg/mL,P ＜ 0.01).The CDDP-resistant cells exhibited significantly increased mitochondrial hTERT expression and mitochondrial membrane potential with increased proportions of CD133 + cells (HepG2:1.44 ± 0.41,HepG2/CDDP:23.15 ± 1.55,P ＜0.01) and EpCAM + cells (HepG2:0.85 ± 0.10,HepG2/CDDP:3.96 ± 0.10,P ＜ 0.01).The resistant cells also showed significantly enhanced colony formation ability and increased expression of OCT-4 protein.Conclusion The translocation of mitochondrial hTERT is significantly increased in CDDP-resistant cells,which possess obvious phenotypes of tumor stem cells.