摘要
目的研究核酸内切酶RNaseH-1对使用端粒的替代延长(ALT)机制来延长端粒的骨肉瘤细胞放射敏感性影响及机制。方法通过慢病毒转染构建过表达RNaseH-1的ALT骨肉瘤细胞U2OS和端粒酶阳性骨肉瘤细胞143B。对转染后的细胞使用CCK8法检测细胞增殖能力,流式细胞仪检测细胞周期。克隆形成实验检测放射敏感性,免疫荧光实验检测细胞DNA损伤(γ-H2AX灶点)情况,蛋白印迹法实验检测相关蛋白表达水平。结果过表达RNaseH-1后U2OS细胞的增殖能力显著降低,且细胞周期阻滞于G1期(均P<0.05)。U2OS细胞中RNaseH-1的过表达使ATM和Chk2的磷酸化水平显著升高、同源重组相关蛋白RAD51和BRCA1的表达显著降低,并导致细胞中DNA损伤水平显著上升、细胞放射敏感性增强(均P<0.05)。而过表达RNaseH-1不对端粒酶阳性的143B细胞产生抑制效应(P>0.05)。结论过表达RNaseH-1抑制了端粒酶阴性骨肉瘤细胞并增强了放射敏感性,其机制可能是RNaseH-1在ALT细胞中抑制同源重组修复并激活ATM信号通路。
Objective To investigate the effect and mechanism of RNaseH-1 on the radiosensitivity of the osteosarcoma cells via the alternative lengthening of telomeres(ALT)mechanism to maintain the telomere length.Methods ALT osteosarcoma cell U2OS and telomerase-positive osteosarcoma cell 143B over-expressing RNaseH-1 were constructed by lentiviral transfection.After cell transfection,cell proliferation and cell cycle were determined using CCK-8 assay and flow cytometry.The effect of RNaseH-1 on the radiosensitivity of osteosarcoma cells was examined by colony formation assay.DNA injury(γ-H2AX foci)was assessed by immunofluorescent assay.The expression levels of related proteins were detected by Western blot.Results The proliferation abilities of U2OS cells were significantly declined following the over-expression of RNaseH-1,and G1 cell cycle arrest was noted(all P<0.05).Over-expression of RNaseH-1 in U2OS cells increased the phosphorylated levels of ATM and Chk2,down-regulated the expression of homologous recombination(HR)-related proteins RAD51 and BRCA1significantly aggravated DNA damage and remarkably enhanced the radiosensitivity(all P<0.05).Over-expression of RNaseH-1 exerted no inhibitory effect upon the telomerase-positive 143B cells(P>0.05).Conclusion RNaseH-1 over-expression suppresses telomerase-negative osteosarcoma cells and enhances the radiosensitivity probably via the role of RNaseH-1 in inhibiting the homologous recombination repair and activating the ATM signaling pathway.
作者
余笑言
王晴晴
王渊
周云峰
Yu Xiaoyan;Wang Qingqing;Wang Yuan;Zhou Yunfeng(Department of Radiation&Medical Oncology,Zhongnan Hospital of Wuhan University,Clinical Cancer Study Center,Key Laboratory of Tumor Biological Behavior of Hubei Province,Wuhan 430071,China)
出处
《中华放射肿瘤学杂志》
CSCD
北大核心
2021年第6期625-630,共6页
Chinese Journal of Radiation Oncology
基金
国家自然科学基金项目资助(81472799)。
