微小RNA217靶向人沉默调节蛋白1对结直肠癌细胞增殖、迁移和侵袭的影响  

Effect of microRNA-217 targeting silent information regulator 1 protein on the proliferation,migration,and invasion of colorectal cancer cells

作　　者：余晓明[1] 赵小乐 裴富雍 林传俊[3] 何梓芸 程菲杨 姜进平[1] 汤守元[1] 罗海平[1] 兰国玉 Yu Xiaoming;Zhao Xiaole;Pei Fuyong;Lin Chuanjun;He Ziyun;Cheng Feiyang;Jiang Jinping;Tang Shouyuan;Luo Haiping;Lan Guoyu(Department of Gastrointestinal Surgery,Huangshi Central Hospital,Affiliated Hospital of Hubei Polytechnic University,Huangshi 435000,China;Department of Gastrointestinal Surgery,Zhongnan Hospital,Clinical Medical Research Center of Peritoneal Cancer of Wuhan,Clinical Cancer Study Center of Hubei Provence,Key Laboratory of Tumor Biological Behavior of Hubei Provence,Wuhan 430071,Wuhan;Department of Clinical Laboratory,Huangshi Central Hospital,Affiliated Hospital of Hubei Polytechnic University,Huangshi 435001,China)

机构地区：[1]黄石市中心医院(湖北理工学院附属医院)胃肠外科,黄石435000 [2]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,武汉430071 [3]黄石市中心医院(湖北理工学院附属医院)检验科,黄石435001

出　　处：《中华实验外科杂志》2023年第7期1326-1329,共4页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金资助项目(82070302、81902018);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-217(miR-217)对结直肠癌细胞增殖、迁移和侵袭的影响途径。方法采用反转录实时荧光定量聚合酶链反应(RT-qPCR)检测人正常结肠上皮细胞NCM460和人结直肠癌细胞SW620、RKO、HT-29、HCT116中miR-217表达水平。将miR-217模拟物(mimic)与阴性对照(mimic NC)分别转染于RKO细胞系, 检测miR-217表达水平。采用细胞计数试剂盒(CCK-8)与平板克隆实验检测细胞增殖活力;采用划痕愈合实验、Transwell实验检测细胞迁移及侵袭能力;采用RT-qPCR与蛋白质印迹法(Western blot)检测沉默信息调节因子1(SIRT1) mRNA与蛋白表达水平。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 miR-217在人结直肠癌细胞系中表达水平降低, 其中RKO细胞系明显低于NCM460细胞系[(0.286±0.022)倍比(1.000±0.090)倍, t=13.360, P<0.01], 差异有统计学意义。RKO细胞系转染miR-217 mimic后, miR-217表达水平高于NC组[(5.276±0.437)倍比(1.000±0.168)倍, t=15.820, P<0.01], 差异有统计学意义。CCK-8实验表明miR-217 mimic组细胞24、48、72 h吸光度值低于NC组[(0.867±0.030)倍比(1.260±0.0222)倍, t=18.440, P<0.01], 差异有统计学意义。平板克隆实验表明miR-217 mimic组细胞集落形成能力低于NC组[(80.570±43.320)倍比(116.600±81.230)倍, t=3.823, P<0.01], 差异有统计学意义。划痕实验及Transwell实验证实miR-217 mimic组细胞迁移及侵袭能力低于NC组[划痕实验:(0.133±0.009)倍比(0.365±0.010)倍, t=29.960, P<0.01;Transwell迁移实验:(157.000±3.000)倍比(248.300±13.580)倍, t=11.380, P<0.01;Transwell侵袭实验:(149.000±7.937)倍比(287.000±5.568)倍, t=24.650, P<0.01], 差异有统计学意义。通过生物信息学分析并筛选出miR-217靶基因人SIRT1, RT-qPCR结果表明miR-217 mimic组细胞中SIRT1 mRNA表达水平低于NC组[(0.682±0.162)倍比(1.000±0.075)倍, t=3.090, P<0.05], 差异有统计学意义。Western blot表明, 转染miR-217 mimic后, 结直�Objective To investigate the pathways by which microRNA-217(miR-217)affects the proliferation,migration,and invasion of colorectal cancer cells.Methods Using reverse transcription re-al-time fluorescence quantitative polymerase chain reaction(RT-qPCR),the expression level of miR-217 was detected in human normal colon epithelial cells NCM460 and human colorectal cancer cells SW620,RKO,HT-29,and HCT116.The miR-217 analog(mimic)and negative control(mimic NC)were trans-fected into RKO cells respectively,and the expression level of miR-217 was detected.Cell proliferation ac-tivity was detected using cell counting kit-8(CCK-8)and plate cloning assays.The ability of cell migration and invasion was detected by scratch healing test and Transwell test.RT-qPCR and Western blotting were used to detect the expression levels of silent information regulator 1(SIRTI)mRNA and protein.The com-parison between the two groups was conducted using t-test,and the comparison between multiple groups was conducted using one-way analysis of variance(ANOVA).Results The expression level of miR-217 decreased in human colorectal cancer cell lines,with RKO cell lines significantly lower than NCM460 cell lines[(0.286±0.022)times vs.(1.000±0.090)times,t=13.360,P<0.01],and the difference was statistically significant.After transfection of miR-217 mimic into the RKO cells,the expression level of miR-217 was higher than that of the NC group[(5.276±0.437)times vs.(1.000±0.168)times,t=15.820,P<0.01],and the difference was statistically significant.The CCK-8 assay showed that the ab-sorbance of cells in the miR-217 mimic group at 24,48,and 72 h was lower than that in the NC group[(0.867±0.030)times vs.(1.260±0.0222)times,t=18.440,P<0.01],and the difference was sta-tistically significant.The plate cloning experiment showed that the colony forming ability of the miR-217 mimic group was lower than that of the NC group[(80.570±43.320)times vs.(116.600±81.230)times,t=3.823,P<0.01],and the difference was statistically significant.Scratch test and Tran

关 键 词：结直肠癌 微小RNA 人沉默调节蛋白1 

分 类 号：R735.34[医药卫生—肿瘤]