微小RNA-17-5p通过抑制转移生长因子β受体2的表达促进结直肠癌细胞增殖、迁移和侵袭  

MicroRNA-17-5p promotes proliferation,migration and invasion of colorectal cancer cells by inhibiting transforming growth factor-βreceptor 2 expression

作　　者：窦婷亭 束琳 陈天亮 赵小乐 方效昌 姜毅楠 冯茂辉[1] 李炫飞 Dou Tingting;Shu Lin;Chen Tianliang;Zhao Xiaole;Fang Xiaochang;Jiang Yinan;Feng Maohui;Li Xuanfei(Department of Gastrointestinal Surgery,Zhongnan Hospital of Wuhan University,Clinical Medical Research Center of Peritoneal Cancer of Wuhan,Clinical Cancer Study Center of Hubei Province,Key Laboratory of Tumor Biological Behavior of Hubei Province,Wuhan 430071,China)

机构地区：[1]武汉大学中南医院胃肠外科武汉市腹膜癌临床医学研究中心湖北省肿瘤医学临床研究中心肿瘤生物学行为湖北省重点实验室,430071

出　　处：《中华实验外科杂志》2022年第10期1911-1914,共4页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金资助项目(82070302、1902018);湖北省卫生健康委资助项目(WJ2021M167);武汉市腹膜癌临床医学研究中心资助项目(2015060911020462);武汉大学中南医院科技创新培育基金资助项目(CXPY2022055)。

摘　　要：目的探讨微小RNA(miR)-17-5p对人结直肠癌细胞增殖、迁移及侵袭功能的影响及其机制。方法培养人结直肠癌细胞RKO、SW620、LOVO、HCT116和人正常结肠上皮细胞NCM460,以上细胞购自美国典型培养物保藏中心。利用反转录实时荧光定量聚合酶链反应(qRT-PCR)检测miR-17-5p在各细胞系内的表达,构建miR-17-5p抑制剂miR-17-5p inhibitor及其阴性对照negative control并转染至RKO细胞,分别设为miR-17-5p抑制剂(miR-17-5p inhibitor)组和阴性对照(NC)组。qRT-PCR法检测miR-17-5p表达。细胞计数试剂盒(CCK-8)检测细胞增殖活性。平板克隆实验检测细胞克隆形成能力。划痕实验检测细胞迁移能力。Transwell实验检测细胞迁移及侵袭能力。qRT-PCR和蛋白印迹法(Western blot)检测转染后细胞内转移生长因子β受体2(TGFBR2)表达水平变化。两组间比较采用t检验,多组间比较采用单因素方差分析(ANOVA)。结果人结直肠癌细胞RKO、SW620、LOVO、HCT116中miR-17-5p的表达倍数均高于人正常结肠上皮细胞NCM460(3.22±0.72、1.99±0.36、2.26±0.51、1.77±0.60比1.07±0.05,F=5.153,P<0.05),差异有统计学意义。miR-17-5p inhibitor组miR-17-5p表达水平低于NC组(0.55±0.07比1.03±0.06,t=9.714,P<0.05),差异有统计学意义。CCK-8实验证实miR-17-5p inhibitor组细胞增殖能力低于NC组(24、48、72、96 h吸光度值分别为(0.21±0.00比0.33±0.03,0.33±0.01比0.54±0.01,0.54±0.02比0.79±0.00,0.83±0.05比1.10±0.08,t=5.041、16.840、17.530、3.977,P<0.05),差异有统计学意义。平板克隆实验提示miR-17-5p inhibitor组细胞克隆形成能力低于NC组[克隆形成数目(327.33±9.53)个比(504.67±10.96)个,t=27.290,P<0.05],差异有统计学意义。划痕及Transwell实验结果显示,miR-17-5p inhibitor组细胞迁移及侵袭能力显著低于NC组[划痕实验细胞迁移率(16.83±1.21)%比(35.70±1.04)%,Transwell迁移实验细胞迁移数目(46.00±3.27)个比(146.33±4.92)个,Transwell侵袭实验细胞Objective To investigate the effect of miR-17-5p on the proliferation,migration and invasive function of human colorectal cancer cells and its molecular mechanism.Methods Human colorectal cancer cells RKO,SW620,LOVO,HCT116 and human normal colonic epithelial cells NCM460 were purchased from American Type Culture Collection and cultured.MiR-17-5p expression was detected in each cell line by reverse transcription quantitative real-time fluorescence polymerase chain reaction(RT-qPCR).The miR-17-5p inhibitor and its negative control were transfected into RKO cells as miR-17-5p inhibitor and negative control(NC)groups,respectively.Cell proliferation activity was measured by cell counting kit-8(CCK-8).The ability of cell clone formation was measured by plate cloning assay.Transwell assay was performed to detect cell migration and invasion activity.RT-qPCR and protein blotting(Western blotting)were performed to detect the change of transforming growth factor-βreceptor 2(TGFBR2)expression in transfected cells.The t-test was used for comparison between two groups,and one-way analysis of variance(ANOVA)was used for comparison between multiple groups.Results The expression fold of miR-17-5p in human colorectal cancer cells RKO,SW620,LOVO and HCT116 was higher than that in human normal colonic epithelial cells NCM460(3.22±0.72,1.99±0.36,2.26±0.51,1.77±0.60 vs.1.07±0.05,F=5.153,P<0.05),the difference was statistically significant.The level of miR-17-5p in miR-17-5p inhibitor group was lower than that in NC group(0.55±0.07 vs.1.03±0.06,t=9.714,P<0.05),the difference was statistically significant.CCK-8 experiment confirmed that the proliferation ability of miR-17-5p inhibitor group was lower than that of NC group[24,48,72,96 h absorbance values were(0.21±0.00 vs.0.33±0.03,0.33±0.01 vs.0.54±0.01,0.54±0.02 vs.0.79±0.00,0.83±0.05 vs.1.10±0.08,t=5.041,16.840,17.530,3.977,P<0.05),the difference were statistically significant.Plate clone assay showed that the clone formation ability of miR-17-5p inhibitor group was lo

关 键 词：结直肠癌 微小RNA 转移生长因子β受体2 增殖 迁移 侵袭 

分 类 号：R735.34[医药卫生—肿瘤]