m6A识别蛋白HuR调控lncRNA TRG-AS1抑制结直肠癌生长的机制研究  

The mechanism of m6A recognition protein HuR inhibiting the growth of colorectal cancer by regulating lncRNA TRG-AS1

作　　者：柴小兵 张利 褚菲菲 吴慧丽 CHAI Xiaobing;ZHANG Li;CHU Feifei;WU Huili(Department of Gastroenterology,Zhengzhou Central Hospital Affiliated to Zhengzhou University,Zhengzhou 450000,China)

机构地区：[1]郑州大学附属郑州中心医院消化内科,450000

出　　处：《天津医药》2023年第2期124-131,共8页Tianjin Medical Journal

基　　金：河南省医学科技攻关计划联合共建项目(LHGJ20210763)。

摘　　要：目的 探讨N6-甲基腺苷(m6A)识别蛋白人类抗原R(HuR)调控长链非编码RNA T细胞受体γ位点反义RNA 1(lncRNA TRG-AS1)对结直肠癌(CRC)生长的影响。方法 比色法检测CRC患者的癌组织、癌旁组织及正常结肠上皮细胞NCM460及CRC细胞HCT116、SW480、LOVO中m6A含量;实时荧光定量PCR(qPCR)检测TRG-AS1表达;Western blot检测HuR蛋白表达。将HCT116细胞分为Ct组、OE-NC组、OE-HuR组、si-NC组、si-HuR组、siHuR+pcDNA组、si-HuR+pcDNA-TRG-AS1组,CCK-8法检测细胞增殖;平板克隆实验检测细胞克隆形成能力;流式细胞术检测细胞凋亡率;划痕愈合实验检测细胞迁移;Transwell检测细胞侵袭;裸鼠体内移植瘤实验观察肿瘤生长情况;采用甲基化RNA免疫共沉淀(MeRIP)检测TRG-AS1上是否存在m6A位点;RNA pull-down实验和RNA免疫共沉淀(RIP)检测TRG-AS1与HuR蛋白的相互作用。结果 在CRC组织和细胞中,HuR蛋白、TRG-AS1高表达,m6A含量降低,且在HCT116细胞中HuR蛋白、TRG-AS1表达最高,m6A含量最低(P<0.05),选择HCT116细胞为研究对象。与si-NC组比较,si-HuR组HuR蛋白、TRG-AS1表达降低,m6A含量升高(P<0.05);与OE-NC组比较,OE-HuR组HuR蛋白、TRG-AS1表达升高,m6A含量降低(P<0.05);与si-HuR组、si-HuR+pcDNA组比较,si-HuR+pcDNATRG-AS1组HuR蛋白、m6A含量变化差异无统计学意义,TRG-AS1表达升高(P<0.05);下调HuR可抑制HCT116细胞增殖、迁移、侵袭及体内移植瘤的生长,促进细胞凋亡,而上调HuR则呈相反趋势;过表达TRG-AS1减弱了沉默HuR对HCT116细胞增殖、划痕愈合率、侵袭、体内移植瘤生长的抑制作用以及对细胞凋亡的促进作用;TRG-AS1上存在m6A位点,且TRG-AS1能与HuR蛋白相互作用。结论 沉默m6A识别蛋白HuR可通过抑制TRG-AS1表达进而抑制HCT116细胞增殖、迁移与侵袭,促进细胞凋亡。Objective To investigate the influence of N6-methyladenosine(m6A) recognition protein human antigen R(HuR) on the growth of colorectal cancer(CRC) by regulating long non-coding RNA T cell receptor gamma locus antisense RNA 1(lncRNA TRG-AS1).Methods The content of m6A in cancer tissue,paracancer tissue and normal colon epithelial cells NCM460 and CRC cells HCT116,SW480 and LOVO were detected by colorimetric method.The expression of TRGAS1 was detected by real-time fluorescence quantitative PCR(qPCR).The expression of HuR protein was detected by Western blot assay.HCT116 cells were divided into the Ct group,the OE-NC group,the OE-HuR group,the si-NC group,the si-HuR group,the si-HuR+pcDNA group and the si-HuR+pcDNA-TRG-AS1 group.CCK-8 assay was applied to detect cell proliferation.Plate cloning assay was used to detect the ability of cells to form clones.Flow cytometry was applied to detect apoptosis.Scratch-healing assay was applied to detect cell migration.Transwell assay was used to detect cell invasion.In vivo tumor xenograft experiment was used to observe tumor growth in nude mice.Methylated RNA immunoprecipitation(MeRIP) was applied to detect the presence of m6A sites on TRG-AS1.RNA co-immunoprecipitation(RIP) and RNA pull-down experiments were used to demonstrate the interaction of TRG-AS1 with HuR protein.Results In CRC tissue and cells,HuR protein and TRG-AS1 were highly expressed,and m6A content was decreased.In HCT116cells,the expression levels of HuR protein and TRG-AS1 were the highest,and the m6A content was the lowest(P <0.05),therefore,HCT116 cells were selected as the research object.Compared with the si-NC group,the expression levels of HuR protein and TRG-AS1 decreased in the si-HuR group,and the m6A content increased(P<0.05).Compared with the OENC group,expression levels of HuR protein and TRG-AS1 increased in the OE-HuR group,and the m6A content decreased(P<0.05).Compared with the si-HuR group and the si-HuR+pcDNA group,there were no significant differences in changes of HuR protein and m6A con

关 键 词：结直肠肿瘤 甲基化 RNA 长链非编码 细胞增殖 人类抗原R N6-甲基腺苷 T细胞受体γ位点反义RNA 1 

分 类 号：R587.24[医药卫生—内分泌]