微小RNA-519d-3p靶向DCAF4L2抑制结直肠癌增殖、迁移和侵袭  

MicroRNA-519d-3p targeting DCAF4L2 inhibits the proliferation,migration and invasion of colorectal cancer

作　　者：方效昌 窦婷亭 束琳 陈天亮 赵小乐 姜毅楠 冯茂辉[1] 李炫飞 Fang Xiaochang;Dou Tingting;Shu Lin;Chen Tianliang;Zhao Xiaole;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1903-1906,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-519d-3p(miR-519d-3p)在结直肠癌(CRC)中的表达及对增殖、迁移及侵袭能力的影响。方法体外培养人结直肠癌细胞系和人正常结肠上皮细胞。将miR-519d-3p模拟物(mimic)分别转染于RKO及SW620,同时设置mimic阴性对照(mimic NC)。采用实时荧光定量聚合酶链反应(RT-qPCR)检测miR-519d-3p表达及DCAF4L2 mRNA表达水平。细胞计数试剂盒(CCK-8)和平板克隆实验检测细胞增殖活性。划痕愈合实验、Transwell迁移和侵袭实验检测细胞迁移及侵袭能力。蛋白质印迹法(Western blot)检测DCAF4L2蛋白表达水平。符合正态分布的计量资料以均数±标准差(±s)表示,采用两样本t检验,多组间比较采用单因素方差分析。结果miR-519d-3p在人结直肠癌细胞系中显著下降,其中RKO和SW620表达倍数明显低于NCM460[RKO:(0.311±0.165)倍比1.000倍,t=7.783,P<0.05;SW620:(0.354±0.079)倍比1.000倍,t=15.72,P<0.01],差异有统计学意义。成功转染mimic过表达miR-519d-3p,过表达组表达倍数高于对照组[RKO:(2.913±0.602)倍比1.000倍,t=5.409,P<0.01;SW620:(3.194±0.427)倍比1.000倍,t=8.765,P<0.001],差异有统计学意义。通过生物学功能实验证实miR-519d-3p表达升高能显著抑制CRC细胞的增殖、迁移和侵袭能力。CCK-8检测结果示,过表达组24、48、72、96 h细胞吸光度值低于对照组(RKO:0.248±0.018比0.184±0.012、0.388±0.007比0.277±0.010、0.658±0.033比0.496±0.008、0.877±0.020比0.681±0.021,t=3.064,P<0.01;SW620:0.213±0.018比0.181±0.050、0.375±0.008比0.274±0.014、0.720±0.009比0.482±0.006、0.978±0.115比0.721±0.032,t=2.432,P<0.01),差异有统计学意义。过表达组平板克隆集落形成个数显著低于对照组[RKO:(262.300±11.320)个比(181.700±2.333)个,t=7.127,P<0.05;SW620:(448.000±33.260)个比(190.300±7.446)个,t=9.387,P<0.05],差异有统计学意义。过表达组划痕愈合率低于对照组[RKO:(39.040±0.812)%比(8.979±0.846)%,t=394.900,P<0.01;SW620:(28.840±0.848)%�Objective To investigate the expression of microRNA-519d-3p(miR-519d-3p)in human colorectal cancer(CRC)and its effect on proliferation,migration and invasion.Methods Human colorectal cancer cell line and human normal colon epithelial cells were cultured in vitro.MiR-519d-3p mimic were transfected into RKO and SW620 respectively,and mimic negative control group(mimic NC group)was set.Quantitative real-time polymerase chain reaction(RT-qPCR)was used to detect the expression of miR-519d-3p and DCAF4L2 mRNA expression level.cell counting kit-8(CCK-8)and colony formation assay were used to detect cell proliferation activity.Wound healing assays,transwell migration and invasion assays were used to detect cell migration and invasion.Western blotting was used to detect DCAF4L2 protein expression level.Two sample t-test was used,and one-way ANOVA was used for comparison among multiple groups.The data were expressed as mean±standard deviation(±s),and the difference was statistically significant when P<0.05.Results MiR-519d-3p was significantly decreased in human colorectal cancer cell lines,and the expression multiples of RKO and SW620 were significantly lower than NCM460[RKO:(0.311±0.165)times vs.1.000 times,t=7.783,P<0.05;SW620:(0.354±0.079)times vs.1.000 times,t=15.72,P<0.01],with statistically significant differences.The overexpression of miR-519d-3p in mimic was successfully transfected.The expression of miR-519d-3p in the overexpression group was higher than that in the control group[RKO:(2.913±0.602)times vs.1.000 times,t=5.409,P<0.01;SW620:(3.194±0.427)times vs.1.000 times,t=8.765,P<0.001],the difference was statistically significant.The biological function experiment confirmed that the increased expression of miR-519d-3p significantly inhibited the proliferation,migration and invasion of CRC cells.CCK-8:the cell absorbance value of the overexpression group at 24,48,72,96 h was lower than that of the control group(RKO:0.248±0.018 vs.0.184±0.012,0.388±0.007 vs.0.277±0.010,0.658±0.033 vs.0.496±0.008,0.877

关 键 词：结直肠癌 微小RNA-519d-3p DCAF4L2 

分 类 号：R735.34[医药卫生—肿瘤]