miR-144-3p靶向调控Nrf2对膀胱癌顺铂敏感性的影响  

Effect of miR-144-3p targeting Nrf2 on cisplatin sensitivity in bladder cancer

作　　者：李瑞晓 董维平 张波 郭博昕 苏瑞平 雷永华 Li Ruixiao;Dong Weiping;Zhang Bo;Guo Boxin;Su Ruiping;Lei Yonghua(Urology Hospital,Xi′an People′s Hospital(Xi′an Fourth Hospital),Xi′an 710199,China;Department of Urology,Shaanxi Armed Police General Hospital,Xi′an 710054,China;Department of Urology,Tangdu Hospital of Air Force Medical University,Xi′an 710038,China)

机构地区：[1]西安市人民医院(西安市第四医院)泌尿肾脏病院,西安710199 [2]陕西武警总医院泌尿外科,西安710054 [3]空军军医大学唐都医院泌尿外科,西安710038

出　　处：《中国医师杂志》2023年第9期1333-1339,共7页Journal of Chinese Physician

基　　金：国家自然科学基金(81872077)。

摘　　要：目的探讨miR-144-3p在膀胱癌顺铂耐药中的作用。方法体外培养膀胱癌细胞T24,将细胞分为空白组(不予处理)、模拟物对照组、miR-144-3p模拟物转染组、抑制剂对照组、miR-144-3p抑制剂转染组。实时荧光定量PCR(qRT-PCR)验证转染效果,MTT法检测各组细胞经顺铂处理后的存活率,蛋白免疫印迹法(Western blot)检测目的蛋白的表达。应用双荧光素报告基因实验验证miR-144-3p与核因子E2相关因子2(Nrf2)的靶向关系,进一步在各组细胞中敲除Nrf2,qRT-PCR和Western blot检测各组细胞中HO-1、Bcl-2和Caspase-3的mRNA和蛋白表达水平。结果与对照组相比,miR-144-3p模拟物转染组膀胱癌细胞对顺铂更敏感,而miR-144-3p抑制剂转染组的作用则相反;miR-144-3p模拟物转染组可有效抑制膀胱癌细胞中Nrf2的mRNA和蛋白表达水平(均P<0.05),而miR-144-3p抑制剂转染组Nrf2的mRNA和蛋白质水平上调(均P<0.05)。miR-144-3p模拟物转染组HO-1和Bcl-2的mRNA和蛋白表达显著下调,Caspase-3表达上调(均P<0.05),而miR-144-3p抑制剂转染组显示出相反的结果。荧光素酶结果证实miR-144-3p可以直接与Nrf2的3′-UTR区域结合,降低Nrf2的mRNA水平。而当Nrf2被敲除时,不管是miR-144-3p模拟物转染组和miR-144-3p抑制剂转染组,HO-1、Bcl-2和Caspase-3的mRNA和蛋白表达水平无明显变化,miR-144-3p失去了调节膀胱癌细胞顺铂敏感性的能力。结论miR-144-3p靶向调控Nrf2对膀胱癌顺铂的敏感性,miR-144-3p有望成为顺铂抗性或难治性膀胱癌治疗的新靶点。Objective To investigate the role of miR-144-3p in cisplatin resistance of bladder cancer.Methods Bladder cancer T24 cells were cultured in vitro and divided into blank group(untreated),mimetic control group,miR-144-3p mimetic transfection group,inhibitor control group,and miR-144-3p inhibitor transfection group.Real time fluorescence quantitative polymerase chain reaction(qRT-PCR)was used to verify the transfection effect,methyl thiazolyl tetrazolium(MTT)method was used to detect the survival rate of cells treated with cisplatin in each group,and Western blot was used to detect the expression of the target protein.The targeting relationship between miR-144-3p and nuclear factor E2 related factor 2(Nrf2)was validated using dual fluorescence reporter gene experiments.Furthermore,Nrf2 was knocked out in each group of cells,and the mRNA and protein expression levels of HO-1,Bcl-2,and Caspase-3 were detected by qRT-PCR and Western blot in each group of cells.Results Compared with the control group,bladder cancer cells in the miR-144-3p mimetic transfection group were more sensitive to cisplatin,while the miR-144-3p inhibitor transfection group had the opposite effect;The miR-144-3p simulant transfection group can effectively inhibit the mRNA and protein expression level of Nrf2 in bladder cancer cells(all P<0.05),while the miR-144-3p inhibitor transfection group can up regulate the mRNA and protein level of Nrf2(all P<0.05).The miR-144-3p mimetic transfection group showed significant downregulation of mRNA and protein expression of HO-1 and Bcl-2,while the expression of Caspase-3 was upregulated(all P<0.05),while the miR-144-3p inhibitor transfection group showed the opposite results.The luciferase results confirmed that miR 1443p can directly bind to the 3′-UTR region of Nrf2,reducing the mRNA level of Nrf2.When Nrf2 was knocked out,whether miR-144-3p mimetic transfection group or miR-144-3p inhibitor transfection group,the mRNA and protein expression levels of HO-1,Bcl-2 and Caspase-3 did not change significantly

关 键 词：膀胱肿瘤 抗药性 肿瘤 miR-144-3p NF-E2相关因子2 

分 类 号：R737.14[医药卫生—肿瘤]