摘要
目的研究RNA干扰抑制UHRF1表达对食管癌细胞系（TE-1）放射敏感性的影响及作用机制。方法以慢病毒感染方法将UHRF1基因的短发夹状RNA（shRNA）转入TE-1细胞，并分为未转染组、转染NC—shRNA组、转染UHRF1-shRNA组，前二者为对照组。采用RT—PCR和蛋白印记法检测转染前后细胞UHRF，的mRNA和蛋白表达，成克隆法、流式细胞术及蛋白印记法检测转染UHRF1-shRNA联合x线照射对TE-1细胞放射敏感性、周期、凋亡及DNA损伤标识蛋白1-H2AX的影响。结果转染UHRF1-shRNA后TE-1细胞UHRF1的mRNA和蛋白表达受抑（0．11、0．96、0．98，F=124．21，P=0．000；0．10、0．89、0．94，F=125．25，P=0．000）。X线照射后转染UHRF1-shRNA组放射增敏比分别为1．53（D。值比）和1．95（Dq值比）。x线照射可引起TE-1细胞G2＋M期阻滞、凋亡率和γ—H2 AX表达增加，与对照组相比转染UHRF1-shRNA组照后24的G：＋M期阻滞显著降低（F=500．15，P=0．000）、凋亡率和γ-H2AX残存量明显升高（F=100．10、61．00，P=0．000、0．000）。结论RNA干扰可有效抑制TE-1细胞UHRF，表达，增强细胞放射敏感性，其增敏机制与周期调控、凋亡及DNA损伤修复有关。
Objective To study the effect of UHRF1 expression inhibition by RNA interference on the radiosensitivity of esophageal cancer cell line TE-1 and its mechanism. Methods Short hairpin RNA （shRNA） targeting UHRF1 gene was introduced into TE-1 cells by lentivector-mediated transfer. The cells were divided into three groups ：non-transfected group, negative control （NC）-shRNA-transfeeted group, and UHRF1 -shRNA-transfected group. The mRNA and protein expression levels of UHRF1 in TE-1 cells were measured by RT-PCR and Western blot before and after transfection. After transfeetion and X-ray radiation, the radiosensitivity of TE-1 cells was evaluated by colony formation assay; the cell cycle and cell apoptosis were determined by flow cytometry; the γ-H2AX （as a marker of DNA damage） level was measured by Western blot. Results After transfeetion with UHRF1-shRNA, the mRNA and protein expression levels of UHRF1 were significantly decreased in TE-1 cells, as compared with those in the NC-shRNA-transfeeted group and non-transfected group （0. 11 vs 0. 96 and 0. 98, F = 124. 21, P = 0. 000 ;0. 10 vs 0. 89 and 0. 94, F = 125.25, P = 0. 000）. The UHRFl-shRNA-transfected group had sensitization enhancement ratios of 1.53（D0 ratio） and 1.95（Dq ratio）. X-ray radiation could cause G2/M arrest and increase apoptotic rate and-γ-H2AX expression in TE-1 cells. Compared with the two control groups, the UHRF1-shRNA- transfected group showed significantly less G2/M arrest （ F = 500. 15, P = 0. 000 ）, a significantly higher apoptotic rate （ F = 100. 10 ,P = 0. 000）, and significantly higher residual 7-H2AX expression （ F = 61.00, P = 0. 000） at 24 hours after X-ray radiation. Conclusions RNA interference can effectively inhibit the UHRF1 expression and enhance the radiosensitivity of TE-1 cells. The mechanism may be related to cell cycle regulation, cell apoptosis, and DNA damage repair.
出处
《中华放射肿瘤学杂志》
CSCD
北大核心
2013年第4期326-329,共4页
Chinese Journal of Radiation Oncology
