内含子源性长链非编码RNA SOS1-IT1对肝癌细胞的调控  

Regulation of hepatocellular carcinoma cells by long non-coding RNA SOS1-IT1

作　　者：付楠楠[1] 刘蕊 刘涛[2] FU Nannan;LIU Rui;LIU Tao(Gastroenterology Department,Tianjin Children′s Hospital,Tianjin 300134,China)

机构地区：[1]天津市儿童医院(天津大学儿童医院)消化科,天津300134 [2]天津市第一中心医院国家卫生健康委员会危重病急救医学重点实验室,天津300192

出　　处：《实用医学杂志》2021年第19期2447-2452,2457,共7页The Journal of Practical Medicine

基　　金：国家自然科学基金项目(编号:81402322);天津市第一中心医院科技基金项目(编号:2020CM01)。

摘　　要：目的探寻内含子来源的长链非编码RNA(lncRNA)SOS1-IT1对肝癌细胞的影响及分子机制。方法用人肝癌细胞系QGY-7703作为细胞模型,CCK-8实验检测细胞活性,EdU实验检测细胞DNA复制活性。生物信息学预测RNA序列中的miR-124潜在结合位点(MRE)。用真核表达质粒pcDNA3.1(+)作为SOS1-IT1及其MRE,以及miR-124的过表达载体。将MRE克隆插入增强型绿色荧光蛋白(EGFP)的编码区下游,构建荧光报告基因质粒。荧光报告基因实验检测miR-124对MRE序列的特异性结合和调控。结果在人肝癌QGY-7703细胞系中,过表达lncRNA SOS1-IT1能够增强细胞的活性[(1.184±0.114)vs.(0.928±0.104),P<0.05]并加速DNA复制[(0.625±0.013)vs.(0.206±0.016),P<0.05],抑制SOS1-IT1则降低细胞活性[(0.648±0.062)vs.(0.870±0.091),P<0.05]并减缓DNA复制[(0.126±0.022)vs.(0.271±0.033),P<0.05]。生物信息学预测发现,SOS1 mRNA及SOS1-IT1分别包含3个和1个潜在的miR-124的MRE。荧光报告基因实验确定,SOS1 mRNA的前2个MRE,以及SOS1-IT1的MRE均为miR-124的有效结合位点。用包含SOS1-MRE的荧光报告基因质粒转染QGY-7703细胞,同时表达SOS1-IT1的MRE能够增强荧光强度[(3.68±0.315)vs.(2.71±0.180),P<0.05]。进一步过表达miR-124后,荧光强度发生回落[(1.09±0.143)vs.(4.04±0.079),P<0.05]。若将任意一个MRE序列突变,荧光强度不再发生变化[(2.57±0.244)vs.(2.71±0.180);(2.66±0.200)vs.(2.88±0.169),均P>0.05]。结论内含子来源的lncRNA SOS1-IT1能够与宿主基因SOS1竞争性结合细胞内源性miR-124,通过竞争性内源RNA(ceRNA)机制促进SOS1的表达,并促进肝癌细胞的活性和DNA复制。ObjectiveTo explore regulation of long non-coding RNA(lnc RNA)SOS1-IT1 on hepatocellularcarcinoma(HCC)cells and the underlying molecular mechanism.MethodsHuman HCC cell line QGY-7703 was served as a cell model.CCK-8 assay was performed to detect cell viability,and Ed U assay was applied todetect DNA replication activity.The potential mi RNA binding sites(mi RNA response elements,MREs)werepredicted by bioinformatics analysis.The eukaryotic expression plasmid pc DNA3.1(+)was used in ectopic expres-sions of SOS1-IT1,SOS1-MRE and mi R-124.The MREs were cloned into the downstream of an enhanced greenfluorescent protein(EGFP)coding region to form fluorescent reporter plasmids.Fluorescent reporter gene assaywas performed to detect the specific binding and regulation of mi R-124 to the MREs.ResultsIn human HCC cellline QGY-7703,overexpression of lnc RNA SOS1-IT1 led to increase cell viability[(1.184±0.114)vs.(0.928±0.104),P<0.05]and DNA replication[(0.625±0.013)vs.(0.206±0.016),P<0.05],while suppression ofSOS1-IT1 resulted in decreased cell viability[(0.648±0.062)vs.(0.870±0.091),P<0.05]and DNA replication[(0.126±0.022)vs.0.271±0.033,P<0.05].Bioinformatics analysis showed that SOS1 m RNA contained threeand SOS1-IT1 contained one potential mi R-124 reaction elements(MREs).Fluorescent reporter gene assay indicatedthat the first two MREs within SOS1 m RNA and the MRE within SOS1-IT1 were the functional binding sites ofmi R-124.QGY-7703 cells were transfected with the fluorescent reporter plasmid containing SOS1-MREs,along with SOS1-IT1-MRE expression plasmid,and an increase in fluorescent intensity was observed[(3.68±0.315)vs.(2.71±0.180),P<0.05].Further overexpression of mi R-124 led to a fallback of the fluorescence intensity[(1.09±0.143)vs.(4.04±0.079),P<0.05].A change in fluorescence intensity did not occur if any of the MREs wasmutated[(2.57±0.244)vs.(2.71±0.180)and(2.66±0.20)vs.(2.88±0.169),P>0.05].Conclusionslnc RNASOS1-IT1 competitively binds endogenous mi R-124 with its host gene SOS1.SOS1-IT1 promo

关 键 词：肝细胞癌 长链非编码RNA 微RNA 竞争性内源RNA 内含子 

分 类 号：R735.3[医药卫生—肿瘤]