微小RNA-6791-5p靶向磷酸呋喃酸性簇分选蛋白2抑制结直肠癌增殖、迁移和侵袭  

MicroRNA-6791-5p targeting phosphofurin acidic cluster sorting protein 2 inhibits the proliferation,migration and invasion of colorectal cancer

作　　者：孙率真 何梓芸 裴富雍 李炫飞 冯茂辉[1] 姜毅楠 程伏林[1] Sun Shuaizhen;He Ziyun;Pei Fuyong;Li Xuanfei;Feng Maohui;Jiang Yinan;Cheng Fulin(Department of Gastrointestinal Surgery,Zhongnan Hospital of Wuhan University,Clinical Medical Research Center of Peritoneal Cancer of Wuhan,Hubei Key Laboratory of Tumor Biological Behaviors,Hubei Province Cancer Clinical Study Center,Zhongnan Hospital of Wuhan University,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、肿瘤生物学行为湖北省重点实验室、湖北省肿瘤医学临床研究中心,武汉430071

出　　处：《中华实验外科杂志》2023年第9期1769-1772,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);吴阶平医学基金会临床科研专项资助项目(320.6750.2023-11-9);武汉大学中南医院科技创新培育基金资助项目(CXPY2022055);武汉大学中南医院医学科技创新平台建设支撑项目资助项目(PTXM2023004,PTXM2023020)。

摘　　要：目的探讨微小RNA(miR)-6791-5p对人结直肠癌细胞增殖、迁移和侵袭功能的影响及其分子机制。方法采集培养人结直肠癌细胞系RKO、SW480、DLD1、HCT116以及人正常结肠上皮细胞NCM460。通过反转录实时荧光定量聚合酶链反应(RT-qPCR)检测miR-6791-5p在各细胞系中的表达水平(表达倍数为0.52±0.04、0.83±0.06、0.60±0.12、0.68±0.07),并构建miR-6791-5p模拟物(miR-6791-5p mimic)及其阴性对照Negative control,并将其转染至RKO细胞。使用RT-qPCR检测miR-6791-5p和PACS2 mRNA的表达水平(表达倍数为0.468±0.032)。利用蛋白质印迹法(Western blot)检测PACS2蛋白的表达水平[RKO:(51.590±6.018)%]。使用两样本t检验进行统计分析,结果以均数±标准差(±s)表示,以P<0.05为差异有统计学意义。结果miR-6791-5p在人结直肠癌细胞系表达水平显著低于人结肠上皮细胞,其中RKO和DLD1下调最明显(RKO组低于460组:0.52±0.04比1.00、DLD1组低于460组0.60±0.12比1.00),差异有统计学意义(RKO:t=17.811,P<0.0001;DLD1:t=5.372,P<0.01)。RKO转染后,mimic组miR-6791-5p表达水平高于mimic NC组(3.28±0.44比1.00),差异有统计学意义(RKO:t=8.764,P<0.01)。细胞计数试剂(CCK-8)检测表明mimic组24、48、72、96 h细胞吸光度值显著低于mimic NC组(24 h吸光度值为0.185±0.004比0.250±0.030,48 h吸光度值为0.275±0.040比0.376±0.020,72 h吸光度值为0.497±0.120比0.657±0.010,96 h吸光度值为0.691±0.004比0.905±0.019),差异有统计学意义(RKO:t=10.821,P<0.01)。平板克隆实验表明mimic组集落形成数低于mimic NC组(115.70±12.51比176.00±19.18),差异有统计学意义(RKO:t=4.581,P<0.05)。划痕愈合实验显示miR-6791-5p mimic组划痕愈合率低于NC组[(14.960±0.848)%比(36.480±1.340)%],差异有统计学意义(t=23.491,P<0.0001)。Transwell迁徙和侵袭实验显示穿透微孔膜的细胞数mimic组低于mimic NC组(迁移数目miR-6791-5p mimic组比NC组42.330±3.512比125.700±7.760,侵袭数目分别为54.33±4Objective To investigate the impact of microRNA(miR)-6791-5p on the proliferation,migration and invasion of human colorectal cancer cells and its underlying molecular mechanism.Methods Human colorectal cancer cell lines RKO,SW480,DLD1,HCT116,and human normal colon epithelial cells NCM460 were cultured and collected.The expression levels of miR-6791-5p in different cell lines were detected using reverse transcription quantitative real-time polymerase chain reaction(RT-qPCR)(relative expression 0.52±0.04,0.83±0.06,0.60±0.12,0.68±0.07).MiR-6791-5p mimic and its negative control(negative control)were constructed and transfected into RKO cells.The expression levels of miR-6791-5p and PACS2 mRNA were measured using RT-qPCR(relative expression 0.468±0.032).The expression level of PACS2 protein was detected using Western blotting[RKO:(51.590±6.018)%].Statistical analysis was performed using two-sample t-test,and the results were presented as mean±standard deviation(±s).A P-value less than 0.05 indicated statistically significant differences.Results MiR-6791-5p expression was significantly lower in human colorectal cancer cell lines compared to human normal colon epithelial cells,with the most prominent downregulation observed in RKO and DLD1 cells(RKO group lower than NCM460 group 0.52±0.04 vs.1.00,DLD1group lower than NCM460 group 0.60±0.12 vs.1.00),showing statistically significant differences(RKO:t=17.811,P<0.0001;DLD1:t=5.372,P<0.01).After transfection in RKO cells,the expression level of miR-6791-5p was higher in the mimic group compared to the mimic NC group(mimic group was higher than the NC group 3.28±0.44 vs.1.00),exhibiting statistical significance(RKO:t=8.764,P<0.01).cell counting kit-8(CCK-8)assay indicated that the A values of the mimic group were significantly lower than those of the mimic NC group at 24,48,72,and 96 h(24 h A value was 0.185±0.004 vs.0.25±0.03,48 h A value was 0.275±0.04 vs.0.376±0.02,72 h A value was 0.497±0.12 vs.0.657±0.01,96h A value was 0.691±0.004 vs.0.905±0.019),sh

关 键 词：结直肠癌 微小RNA 磷酸呋喃酸性簇分选蛋白2 

分 类 号：R735.34[医药卫生—肿瘤]