微小RNA-29b通过靶向调节Tribbles假激酶2的表达促进结直肠肿瘤的衰老  

MicroRNA-29b promotes cellular senescence of colorectal tumors by targeting tribbles pseudokinase 2

作　　者：侯振林 王桂华 胡俊波[1] 王晶[3] 孙黎[4] Hou Zhenlin;Wang Guihua;Hu Junbo;Wang Jing;Sun Li(Cancer Research Institute,Tongji Hospital,Huazhong University of Science and Technology,Wuhan 430030,China;Colorectal Surgery,Sun Yat-sen University Cancer Center,State Key Laboratory of Oncology in South China,Collaborative Innovation Center for Cancer Medicine,Guangzhou 510060,China;Department of Immunology,Tongji Medical College,Huazhong University of Science and Technology,Wuhan 430030,China;Department of Oncology,Tongji Hospital,Huazhong University of Science and Technology,Wuhan 430030,China)

机构地区：[1]华中科技大学同济医学院附属同济医院分子医学中心,武汉430030 [2]中山大学肿瘤防治中心,华南肿瘤学国家重点实验室肿瘤医学协同创新中心结直肠外科,广州510060 [3]华中科技大学同济医学院基础医学院免疫学系,武汉430030 [4]华中科技大学同济医学院附属同济医院肿瘤中心,武汉430030

出　　处：《中华实验外科杂志》2019年第12期2203-2206,共4页Chinese Journal of Experimental Surgery

摘　　要：目的探讨结直肠肿瘤中微小RNA(miRNA,miR)-29b对Tribbles假激酶2(TRIB2)调节的机制.方法利用网上在线数据库分析潜在结合TRIB2的3'端非编码区(3'UTR)的miRNA,选取其中评分最高的miR-29b进行研究;利用真核细胞转染技术,干扰结直肠肿瘤细胞中miR-29b的表达;蛋白质印迹法(Western blot)以及实时定量聚合酶链反应(Real-time PCR)检测各组细胞中TRIB2的蛋白及mRNA表达差异;利用双荧光素酶报告基因实验确认miR-29b结合在TRIB2-3'UTR上的具体位置;β-半乳糖苷酶染色法检测miR-29b对结直肠肿瘤细胞衰老的影响.统计学分析均应用SPSS 24.0软件进行,两组间统计学差异通过双尾t检验进行分析.结果在结肠癌细胞株中过表达miR-29b后,与对照组比较,TRIB2的mRNA水平下降至(63.468±4.154)%和(43.145±7.523)%(t=5.351,P<0.01和t=6.074,P<0.01);转入针对miR-29b的inhibitor后,TRIB2的mRNA水平则升高至(205.033±27.070)%和(233.353±18.649)%(t=4.663,P<0.01和t=9.411,P<0.01).与上述结果一致,转入miR-29b则抑制,inhibitor则上调TRIB2的蛋白水平.而在结肠癌细胞中过表达miR-29b后,野生型TRIB2-3'UTR质粒的荧光素酶活性分别下降至(53.531±3.787)%和(45.051±1.728)%(t=6.928,P<0.01和t=15.570,P<0.01),而突变型TRIB2-3'UTR质粒的荧光素酶活性只有轻微下降[下降至(87.366±3.825)%和(92.839±3.171)%,t=5.578,P<0.01和t=2.245,P>0.05].过表达miR-29b后,结肠癌细胞中衰老的细胞比例从41.473%升高至62.085%(χ^2=19.731,P<0.01),同时过表达TRIB2后,则下降至46.866%(χ^2=12.031,P<0.01).结论miR-29b可靶向调控TRIB2的表达,促进结直肠肿瘤的衰老.Objective To investigate the mechanism of microRNA(miRNA,miR)-29b regulation of tribbles pseudokinase 2(TRIB2)in colorectal tumors.Methods The online database was used to analyze the miRNAs that bind to the 3'Untranslated Region(UTR)of TRIB2,and the highest scored miR-29b was selected for study.The eukaryotic cell transfection technique was used to interfere with the expression of miR-29b in colorectal tumor cells;Western blotting and real-time quantitative polymerase chain reaction(Real-time PCR)was used to detect the difference in protein and mRNA expression of TRIB2;the position of miR-29b binding on TRIB2-3'untranslated regions(3'UTR)was confirmed by luciferase reporter gene assay;p-galactosidase staining was used to detect effects of miR-29b on colorectal cancer cell senescence.All data were quantified as Mean±SD.Two-tailed Student's Z-test was used to of TRIB2 decreased to(63.468±4.154)%and(43.145±7.523)%(z=5.351,P<0.01 and t=6.074,P<0.01)compared to the control group.In the cells transferee!to the inhibitor targeted miR-29b,the mRNA level of TR1B2 increased to(205.033±27.070)%and(233.353±18.649)%(t=4.663,P<0.01 and t=9.411,P<0.01).Consistent with the above results,miR-29b suppressed,and the inhibitor up-regulated the protein level of TRIB2.After overexpression of miR-29b in colon cancer cells SW48 and SW480,the luciferase activity of the wild-type TRIB2-3'UTR plasmid decreased to(53.531±3.787)%and(45.051±1.728)%,respectively(t=6.928,15.570,P<0.01),while the luciferase activity of the mutant TRIB2-3'UTR plasmid decreased slightly(decreased to(87.366±3.825)%and(92.839±3.171)%,t=5.578,P<0.01 and t=2.245,P>0.05).In the cancer cells overexpressing of miR-29b,the proportion of senescent cells increased from 41.473%to 62.085%(χ^2=19.731,P<0.01),and overexpression of TRIB2 reduced the proportion to 46.866%(χ^2=12.031,P<0.01).Conclusion miR-29b can inhibits TRIB2 expression and promote the senescence of colorectal tumors,and it can be used as a potential therapeutic target for colorectal cancer.

关 键 词：微小RNA-29b Tribbles假激酶2 结直肠癌 细胞衰老 

分 类 号：R73[医药卫生—肿瘤]