lncRNA ANK3DT调控CtIP转录影响宫颈癌细胞的放射敏感性  

lncRNA ANK3DT affects radiosensitivity of cervical cancer cells by regulating CtIP transcription

作　　者：赵文娜 陈子晗 陈富强 杜杰 周美娟 Zhao Wenna;Chen Zihan;Chen Fuqiang;Du Jie;Zhou Meijuan(Department of Radiation Medicine,Guangdong Provincial Key Laboratory of Tropical Disease Research,School of Public Health,Southern Medical University,Guangzhou 510515,China)

机构地区：[1]南方医科大学公共卫生学院放射医学系、广东省热带病研究重点实验室,广州510515

出　　处：《中华放射医学与防护杂志》2025年第3期170-177,共8页Chinese Journal of Radiological Medicine and Protection

基　　金：国家自然科学基金(81903256)。

摘　　要：目的探讨长链非编码RNA ANK3DT(lncRNA ANK3DT)对宫颈癌HeLa细胞DNA损伤修复能力和放射敏感性的影响。方法通过前期构建的基于CRISPR/Cas9系统针对同源重组(HR)和非同源末端连接(NHEJ)修复的定量检测体系,分析lncRNA ANK3DT对人宫颈癌HeLa细胞DNA双链断裂(DSB)修复效率的影响。采用克隆形成实验、流式细胞术、免疫荧光检测下调lncRNA ANK3DT对细胞放射敏感性、X射线照射后细胞凋亡和G 2/M期阻滞、DSB修复的影响。利用Western blot、荧光定量PCR(qPCR)、双荧光素酶报告质粒检测lncRNA ANK3DT对HR修复相关蛋白CtIP的表达和转录的调控作用。结果下调lncRNA ANK3DT显著抑制HR修复,对NHEJ修复无影响;X射线照射后24、48、72 h,HeLa细胞中lncRNA ANK3DT表达显著升高(t=-23.39、-88.83、-52.42,P<0.05);下调lncRNA ANK3DT抑制X射线诱导的DSB修复,提高凋亡水平(t=-14.63,P<0.05),延长G 2/M期阻滞(t=-19.50,P<0.05),增加HeLa细胞的放射敏感性(放射增敏比=1.21);下调lncRNA ANK3DT抑制CtIP启动子活性,降低其mRNA和蛋白的表达。结论lncRNA ANK3DT通过调控CtIP转录影响HR修复,下调lncRNA ANK3DT表达通过抑制电离辐射后DSB修复进而提高HeLa细胞放射敏感性。Objective To explore the effect of long noncoding RNA ANK3DT(lncRNA ANK3DT)on DNA damage repair ability and radiosensitivity of cervical cancer HeLa cells.Methods The effect of lncRNA ANK3DT on the repair efficiency of DNA double-strand breaks(DSBs)was analyzed by the pre-constructed quantitative assay system based on the CRISPR/Cas9 system targeting homologous recombination(HR)and non-homologous end-joining(NHEJ)repair.Clone formation assay,flow cytometry,and immunofluorescence were used to detect the effects of down-regulation of lncRNA ANK3DT on cellular radiosensitivity,apoptosis and G 2/M phase arrest,and DSB repair after X-ray irradiation.Western blot,qPCR,and dual fluorokinase reporter gene plasmid was used to detect the effects of lncRNA ANK3DT on the HR repair-related protein CtIP expression and transcriptional regulation.Results Down-regulation of lncRNA ANK3DT significantly inhibited HR repair and had no effect on NHEJ repair.The expression of lncRNA ANK3DT was significantly increased in HeLa cells at 24,48,72 h after X-ray irradiation(t=-23.39,-88.83,-52.42,P<0.05).Down-regulation of lncRNA ANK3DT inhibited X-ray-induced DSB repair and increased apoptosis levels(t=-14.63,P<0.05),prolonged G 2/M phase block(t=-19.50,P<0.05),increased the radiosensitivity of HeLa cells(radiosensitization ratio=1.21),inhibited the CtIP promoter activity and decreased its mRNA and protein expression.Conclusionslnc RNA ANK3DT affects HR repair by regulating CtIP transcription,and down-regulation of lncRNA ANK3DT increases the radiosensitivity of HeLa cells by inhibiting DSB repair after ionizing radiation.

关 键 词：长链非编码RNA 放射敏感性 DNA损伤 同源重组修复 

分 类 号：R73[医药卫生—肿瘤]