miRNA-34a过表达对4种人宫颈癌细胞放射敏感性研究

目的 研究miRNA-34a过表达对4种人宫颈癌细胞放射敏感性的影响。方法 运用脂质体2000转染试剂盒将pGenesil-1-miRNA-34a表达质粒转染宫颈癌细胞系C33A、HeLa、SiHa和CaSki,通过G418筛选构建过表达miRNA-34a宫颈癌细胞系(miRNA-34a/C33A、miRNA-34a/HeLa、miRNA-34a/SiHa和miRNA-34a/CaSki)。细胞经不同剂量⁶⁰Coγ射线照射;运用TaqManRT-PCR和蛋白印迹法测定细胞中miRNA-34a表达;运用MTT法和克隆形成实验分别测定细胞增殖抑制率和克隆形成能力,流式细胞仪测定细胞凋亡率和蛋白印迹法测定细胞凋亡相关蛋白表达变化。结果 宫颈癌细胞系中miRNA-34a表达量显著高于阴性对照组(P<0.05),过表达miRNA-34a宫颈癌细胞增殖抑制率显著高于阴性对照组(P<0.05),细胞克隆形成率显著低于阴性对照组(P<0.05)。流式细胞仪结果显示过表达miRNA-34a宫颈癌细胞凋亡率显著高于对照组(P<0.05)。蛋白印迹法结果显示4 Gy+过表达miRNA-34a细胞中cleaved caspase 9和cleaved PARP蛋白表达量高于过表达miRNA-34a细胞。结论 本研究成功构建miRNA-34a过表达宫颈癌细胞系,miRNA-34a过表达增强宫颈癌细胞放射敏感性,其机制与激活细胞凋亡通路有关。

Effects of microRNA-34a overexpression on radiosensitivity of four human cervical cancer cell lines

Objective To investigate the effects of miRNA-34a overexpression on the radiosensitivity of four human cervical cancer cell lines. Methods Cervical cancer lines, C33A, HeLa, SiHa, and CaSki, were transfected with pGenesil-1-miRNA-34a plasmid using the liposome 2000 kit. Cervical cancer lines overexpressing miRNA-34a ( miRNA-34a/C33A, miRNA-34a/HeLa, miRNA-34a/SiHa, and miRNA-34a/CaSki) were constructed by G418 selection. These cells were exposed to different doses of 60 Coγ-ray. The expression of miRNA-34a was determined using TaqMan real-time PCR and Western blot. The cell growth inhibition rate and colony formation ability were measured by MTT assay and colony formation assay, respectively. Apoptosis and expression of apoptosis-related proteins were evaluated using flow cytometry and Western blot, respectively. Results The expression of miRNA-34a was significantly higher in positive cervical cancer cell lines than in the negative control groups ( P<0. 05 ) . The cervical cancer cell lines overexpressing miRNA-34a had significantly elevated growth inhibition rates and significantly weaker colony formation ability than the negative control groups ( both P<0. 05) . The results of flow cytometry showed that cervical cancer cell lines overexpressing miRNA-34a had significantly higher apoptosis rates than the negative control groups ( P<0. 05 ) . The results of Western blot indicated that cervical cancer lines overexpressing miRNA-34a had increased expression of cleaved caspase 9 and cleaved PARP proteins after exposure to 4 Gy+ radiation. Conclusions Cervical cancer lines overexpressing miRNA-34a are successfully constructed in this study. Overexpression of miRNA-34a enhances the radiosensitivity of cervical cancer cells. The mechanism is related to the activation of apoptosis pathway.