摘要
目的探讨短发卡RNA(shRNA)干扰沉默骨桥蛋白(osteopontin,OPN)基因对宫颈癌细胞株HeLa化疗敏感性的影响及其可能机制。方法 OPNshRNA真核表达质粒pGCsi3.0在脂质体介导下转染HeLa细胞(shOPN组),以转染空载质粒的细胞(shNon组)和未转染细胞(Con组)作为对照。分别用0、0.5、1、2μg/mL顺铂和0、50、100、500nmol/L紫杉醇处理各组细胞24h,流式细胞术检测细胞凋亡。蛋白质印迹分析检测下调OPN表达后细胞凋亡相关蛋白cleaved caspase-3、Bcl-xL、Bcl-2和Bax表达的变化。结果 2μg/mL顺铂处理后shOPN组细胞凋亡率为(44.53±2.78)%,与shNon组[(15.34±2.18)%]和Con组[(15.37±1.03)%]比较差异有统计学意义(P<0.05)。500nmol/L紫杉醇处理后shOPN组细胞凋亡率为(51.46±1.49)%,与shNon组[(19.16±1.87)%]和Con组[(17.03±2.37)%]比较差异有统计学意义(P<0.05)。下调OPN表达后顺铂对cleaved caspase-3的活化作用增强(P<0.01),同时抗凋亡蛋白Bcl-xL、Bcl-2的表达下调,促凋亡蛋白Bax表达上调。结论 OPN沉默后可通过促进宫颈癌HeLa细胞的凋亡进而增强细胞对化疗药物的敏感性,OPN的RNA干扰可能成为一种新的治疗策略应用于宫颈癌的新辅助化疗。
Objective To investigate the effect of silencing osteopontin （OPN） expression by short-hairpin RNA （shRNA） interference on chemotherapy sensitivity of cervical cancer HeLa ceils and the possible mechanism. Methods HeLa cells were transfected with eukaryotic expression vector pGCsi3. 0 carrying OPN shRNA via liposome （shOPN group）. Untransfected HeLa cells （Con group） and those transfected with empty plasmids （shNon group） served as controls. HeLa cells in all the groups were treated with different concentrations of cisplatin （0, 0.5, 1, and 2 μg/mL） and paclitaxel （0, 50, 100, and 500 nmol/L） for 24 h, respectively the apoptosis in apoptosis related protein cleaved caspase-3, Bcl-xL, Bcl-2, and Hela cells was analyzed by flow cytometry. The expressions of Bax were examined by Western blotting analysis. Results The apoptotic rate in shOPN group （[44.53±2.78] % ） was significantly higher than those in shNon group （[15.34 ± 2.18] %） and Con group （[15. 37±1.03]G） after treatment with 2 /ag/mL cisplatin （P〈0.05）3 and significant difference was also found between the apoptotie rates after treatment with 500 nmol/L paclitaxel （shOPN group： [-51. 46 ± 1. 49]G, shNon group： [19. 164-1.87]%, Con group： [-17. 03± 2. 37]% P〈0. 05）. Down-regulating OPN expression significantly enhanced the cisplatin-indueed activation of cleaved caspase-3 （P〈0.01）, and it also resulted in inhibition of Bcl-2/Bcl-xL expression and up- regulation of Bax expression. Conclusion OPN silencing can sensitize cervical cancer HeLa cells to chemotherapeutic agents by promoting HeLa cells apoptosis. RNA interference mediated depletion of OPN may be a promising strategy for the new adjuvant chemotherapy treatment of cervical cancer.
出处
《第二军医大学学报》
CAS
CSCD
北大核心
2013年第12期1299-1303,共5页
Academic Journal of Second Military Medical University
关键词
宫颈肿瘤
骨桥蛋白
RNA干扰
凋亡调节蛋白质类
uterine cervical neoplasms
osteopontin
RNA interference
apoptosis regulatory proteins
