siRNA干扰Geminin基因对脑胶质瘤放疗敏感性的影响  

Effect of siRNA targetedly interfering Geminin gene on radiotherapy sensitivity of glioma

作　　者：贺维 潘鹭翔 韩宇晨 邹彦琦 顾锦涛 贾博 刘骁 曹正聪 姜雨然 张阔 张旺倩 王舒宁 李萌 张英起 郝强 HE Wei;PAN Luxiang;HAN Yuchen;ZOU Yanqi;GU Jintao;JIA Bo;LIU Xiao;CAO Zhengcong;JIANG Yuran;ZHANG Kuo;ZHANG Wangqian;WANG Shuning;LI Meng;ZHANG Yingqi;HAO Qiang(College of Life Science,Northwest University,Xi’an 710069,China;Department of Bio-pharmaceuticals,School of Pharmacy,Air Force Medical University;School of Basic Medicine,Air Force Medical University)

机构地区：[1]西北大学生命科学学院,西安710069 [2]空军军医大学药学系生物制药学教研室 [3]空军军医大学基础医学院

出　　处：《山西医科大学学报》2023年第1期17-23,共7页Journal of Shanxi Medical University

基　　金：国家自然科学基金项目(82072658)。

摘　　要：目的观察敲低Geminin对人脑胶质瘤细胞放疗敏感性的影响。方法利用GEPIA和CGGA数据库分析Geminin在脑胶质瘤组织中的表达及与生存期的相关性。实验分为两组:si-GL2组(对照组)和si-Gem组(干扰组),设计siRNA干扰序列(si-Gem)和阴性对照序列(si-GL2),分别转染脑胶质瘤细胞LN229和U87,qRT-PCR和Western blot检测转染效率及蛋白表达水平。流式细胞术检测碘化丙啶(PI)染色后DNA含量分布,进而分析敲低Geminin对DNA再复制的影响。通过AnnexinⅤ-FITC/PI法,用流式细胞术检测敲低Geminin对细胞凋亡的影响。CCK-8细胞增殖实验检测敲低Geminin对胶质瘤细胞LN229和U87放疗敏感性的影响。Western blot检测Geminin相关分子Cdt1和DNA相关损伤蛋白γ-H2AX的表达水平。结果脑胶质瘤组织中Geminin的表达水平高于癌旁正常组织,且Geminin表达越高,脑胶质瘤患者预后越差(P<0.05)。与si-GL2组相比,si-Gem组Geminin在LN229和U87细胞中的表达降低(P<0.05)。与si-GL2组相比,si-Gem组敲低Geminin可诱导脑胶质瘤细胞出现DNA再复制表型、细胞凋亡比例显著增加(P<0.05),脑胶质瘤细胞对放疗敏感性增强(P<0.05)。与si-GL2组相比,si-Gem组Geminin相关分子Cdt1蛋白表达水平降低,γ-H2AX的蛋白表达水平升高。结论敲低Geminin可以诱导脑胶质瘤细胞出现DNA再复制表型,造成DNA复制压力,并引起自发性细胞周期阻滞及细胞凋亡增多,进而增强脑胶质瘤细胞放疗敏感性,Geminin有望成为脑胶质瘤治疗的新靶点。Objective To observe the effect of siRNA interfering Geminin gene expression on the radiosensitivity of human glioma cells.Methods GEPIA and CGGA databases were used to analyze the expression of Geminin in glioma tissues and its correlation with survival.The experiment was divided into two groups:control group(si-GL2)and interference group(si-Gem).The siRNA interference sequence(si-Gem)and the negative control sequence(si-GL2)were designed to transfect LN229 and U87 glioma cells,respectively.The transfection efficiency and the protein expression level were detected by qRT-PCR and Western blot.Flow cytometry was used to detect the DNA content distribution after propidium iodide(PI)staining,and then the effect of Geminin knockdown on DNA re-replication was analyzed.Flow cytometry was used to detect the effect of Geminin knockdown on apoptosis by AnnexinⅤ-FITC/PI.The effect of Geminin knockdown on radiotherapy sensitivity of glioma cells LN229 and U87 was detected by CCK-8 cell proliferation assay.The expression levels of Geminin-associated molecule Cdt1 and DNA-associated damage proteinγ-H2AX were detected by Western blot.Results The expression level of Geminin in glioma tissues was higher than that in adjacent normal tissues,and the higher the expression of Geminin,the worse the prognosis of glioma patients(P<0.05).Compared with control group,the expression of Geminin in LN229 and U87 cells in interference group was decreased(P<0.05).Compared with control group,Geminin knockdown induced DNA replication phenotype in interference group,and the apoptosis rate of glioma cells was significantly increased(P<0.05),and the sensitivity of glioma cells to radiotherapy was enhanced(P<0.05).Compared with control group,the expression level of Geminin-related molecule Cdt1 protein in interference group was decreased,and the protein expression level ofγ-H2AX was increased.Conclusion Geminin knockdown can induce DNA re-replication phenotype in glioma cells,resulting in DNA replication stress,spontaneous cell cycle arrest and

关 键 词：脑胶质瘤 GEMININ siRNA DNA复制 细胞周期 细胞凋亡 CDT1 放疗敏感性 

分 类 号：R739.41[医药卫生—肿瘤]