UHRF1对人乳腺癌细胞MDA-MB-231辐射敏感性的影响及作用机制

Effect of UHRF1 on radiosensitivity of human breast cancer cells to X-rays and its mechanisms

目的 了解基因UHRF1的不同表达水平对乳腺癌细胞MDA-MB-231辐射敏感性的影响及潜在的作用机制.方法 利用克隆形成实验观察细胞存活;流式细胞术测定细胞周期;利用DNA片段分析和Annexin V试剂盒测定细胞凋亡;Western blot测定蛋白表达变化;利用经典的染色体分析,观察染色体畸变（着丝粒环和双着丝粒）.结果 与对照相比较,UHRF1转染可将D0值由2.08 Gy提高至3.17 Gy,即降低MDA-MB-231细胞对X射线的辐射敏感性.利用siRNA抑制UHRF1的表达,可将X射线照射后细胞的生存率由41%降低至17%,即增强了细胞的辐射敏感性.UHRF1的高表达,可诱导G2/M期阻滞,抑制细胞凋亡,下调促凋亡蛋白Bax,上调DNA损伤修复蛋白Ku70和Ku80的表达水平,而且,抑制X射线诱导的染色体畸变.结论 UHRF1的高表达可以抑制细胞凋亡,促进DNA损伤修复.因此,通过抑制UHRF1的表达,可作为临床提高乳腺癌放疗疗效的新靶标.

Objective To investigate the effect of UHRF1 on the radiosensitivity to x-ray in human breast cancer MDA-MB-231 cells and the underlying mechanisms. Methods Cell survival was determined by colony formation assay. Cell cycle distribution was measured by flow cytometry. The apoptosis was evaluated by DNA fragmentation assay and Annexin V apoptosis detection kit. Protein expression was analyzed by Western blot, and chromosome aberration （centric rings and dicentrics） were observed by conventional chromosome analysis. Results A significant decrease of radiosensitivity to X-rays was observed in MDA-MB-231 cells transfected with a full-length of human UHRF1 cDNA （ MDA-MB231/UHRF1 ） compared with the control cells （ MDA-MB-231/parental and MDA-MB-231/Neo）, and the D0 value increased from 2.08 to 3. 17 Gy after UHRF1 transfection. In contrast, a decreased expression of UHRF1 by a specific UHRF1-siRNA significantly decreased cell survival from 41% to 17%. The UHRF1-mediated radioresistance was correlated with a G2/M arrest, a decreased apoptosis rate, a down-regulation of the pro-apoptotic protein Bax and up-regulation of the DNA damage repair proteins Ku70 and Ku80.Furthermore, chromosomal aberrations （centric rings and dicentrics） by X-ray were less in MDA-MB-231/UHRF1 than those in MDA-MB-231/parental cells and MDA-MB-231/Neo control cells. Conclusion UHRF1 may be a new target in the radiotherapy of breast cancer via affecting apoptosis and DNA damage repair.