miR-22通过靶向抑制DDIT4促进糖尿病肾病肾小管上皮细胞DNA损伤  

miR-22 promotes DNA damage of renal tubular epithelial cells with dia⁃betic kidney disease by inhibiting DDIT4

作　　者：梁丹 赵思琪 李志阳 肖雅文 丁菁[1] 肖瑛[1] 郭兵[1] LIANG Dan;ZHAO Si-qi;LI Zhi-yang;XIAO Ya-wen;DING Jing;XIAO Ying;GUO Bing(Guizhou Provincial Key Laboratory of Pathogenesis&Drug Research on Common Chronic Diseases,Department of Patho-physiology,Guizhou Medical University,Guiyang 550025,China;Qinhuangdao Military Hospital,Qinhuangdao 066000,China)

机构地区：[1]贵州省常见慢性疾病发病机制及药物研究重点实验室,贵州医科大学病理生理学教研室,贵州贵阳550025 [2]秦皇岛军工医院,河北秦皇岛066000

出　　处：《中国病理生理杂志》2021年第11期1933-1941,共9页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.81860656);贵州省科技厅(黔科合基础-ZK[2021]重点010号);贵州省教育厅青年科技人才成长项目(黔教合KY字[2017]165号);贵州省科技厅计划项目(黔科合平台人才[2019]5081号)。

摘　　要：目的:探究在糖尿病肾病(DKD)中微小RNA-22(miR-22)是否能靶向抑制DNA损伤诱导转录物4(DDIT4)的表达,进而通过毛细血管扩张性共济失调突变蛋白(ATM)-细胞周期检查点激酶2(Chk2)通路促进DNA损伤。方法:(1)C57BL小鼠建立糖尿病(DM)模型,于16周后处死,分析其生化指标并对肾脏组织做HE染色,Western blot检测肾脏组织中p-ATM、p-Chk2和DDIT4蛋白水平;(2)分别用高糖(30 mmol/L葡萄糖)和正常糖(5.5 mmol/L葡萄糖)培养基培养大鼠肾小管上皮细胞NRK-52E,彗星实验检测DNA双链断裂程度,流式细胞术检测高糖对细胞周期的影响,Western blot检测细胞中p-ATM、p-Chk2和DDIT4蛋白水平;(3)构建DDIT4过表达质粒,转染NRK-52E细胞后通过彗星实验检测DDIT4过表达对DNA双链断裂的影响,并构建miR-22模拟物(mimics)和抑制物(inhibitor),分别转染miR-22 mimics和inhibitor,通过Western blot检测细胞中p-ATM、p-Chk2和DDIT4蛋白水平。结果:(1)DM组小鼠肾组织中p-ATM和p-Chk2蛋白水平显著增高,DDIT4蛋白水平显著下降(P<0.05)。(2)高糖培养的NRK-52E细胞比正常糖培养的细胞彗尾更长,p-ATM和p-Chk2蛋白水平显著增高,DDIT4蛋白水平显著下降(P<0.05)。(3)高糖培养的NRK-52E细胞过表达DDIT4后,p-ATM和p-Chk2蛋白水平显著降低,细胞彗尾变短;转染miR-22 mimics后,p-ATM和p-Chk2蛋白水平显著增高,DDIT4蛋白水平显著下降;转染miR-22 inhibitor后,p-ATM和p-Chk2蛋白水平显著下降,DDIT4蛋白水平显著增高(P<0.05)。结论:高糖可导致DNA损伤加重,其机制可能为miR-22靶向抑制DDIT4表达,诱导肾小管上皮细胞发生DNA损伤,从而促进DKD的发生发展。AIM:To explore whether microRNA-22(miR-22)targets DNA damage-inducible transcript 4(DDIT4)in diabetic kidney disease(DKD)and inhibits DNA damage repair through ataxia telangiectasia mutated(ATM)-checkpoint kinase 2(Chk2)signaling pathway.METHODS:(1)The diabetes mellitus(DM)model was estab-lished in C57BL mice,and the mice were sacrificed after 16 weeks.The biochemical indexes were analyzed and the mor-phological changes of renal tissues were observed.The protein levels of p-ATM,p-Chk2 and DDIT4 in renal tissues were detected by Western blot.(2)Rat renal tubular epithelial NRK-52E cells were cultured with high-glucose(30 mmol/L glu-cose)and normal-glucose(5.5 mmol/L glucose)media.The degree of double-strand breaks was detected by comet assay,and the cell cycle was analyzed by flow cytometry.The protein levels of p-ATM,p-Chk2 and DDIT4 in the cells were de termined by Western blot.(3)DDIT4 over-expression plasmid was constructed,and the effect of DDIT4 over-expression on DNA double-strand breaks was detected by comet assay after transfection.The miR-22 mimics and inhibitor were also constructed.The protein levels of p-ATM,p-Chk2 and DDIT4 in the cells were determined by Western blot.RESULTS:(1)The protein levels of p-ATM and p-Chk2 in the renal tissue of DM mice were increased,while the protein level of DDIT4 was decreased(P<0.05).(2)The length of the cell comet tail in the NRK-52E cells with high-glucose culture were longer than those in normal-glucose culture,the protein levels of p-ATM and p-Chk2 were increased,and the protein level of DDIT4 was decreased.(3)The protein levels of p-ATM and p-Chk2 were decreased and the length of comet tail in NRK-52E cells became shorter after DDIT4 over-expression.The protein levels of p-ATM and p-Chk2 were increased and the protein level of DDIT4 was decreased in the NRK-52E cells transfected with miR-22 mimics in high-glucose culture.The protein levels of p-ATM and p-Chk2 were decreased and the protein level of DDIT4 was increased in the NRK-52E cells transfected with miR-22

关 键 词：DNA损伤 微小RNA-22 DNA损伤诱导转录物4 糖尿病肾病 ATM-Chk2信号通路 

分 类 号：R587.2[医药卫生—内分泌] R363.2[医药卫生—内科学]