GRSF1介导miR-346上调hTERT表达的作用与机制研究

发布时间：2018-04-30 04:12

本文选题：microRNA + mi　；参考：《天津医科大学》2015年博士论文

【摘要】：【目的】微小RNA(microRNA,miRNA)是近十几年来研究最广泛、最明确的一类大小约22nt、内源性的小非编码RNA分子。其经典的作用机制是成熟的miRNA组装进入RNA诱导的沉默复合体(RNA-induced silencing complex,RISC),种子序列依据与靶基因mRNA非翻译区的配对程度决定mRNA降解还是翻译抑制,从而在转录后水平调节基因的表达。但是,最近的研究发现,miRNA还可以在转录和转录后水平上调基因的表达,这依赖于miRNA与靶mRNA序列的特异性相互作用,并且与miRNA核糖核蛋白复合体(microribonucleoprotein,microRNP)的具体成分及细胞的功能状态密切相关。另外,miRNA参与的调控网络是复杂的,既可以通过一个miRNA调控多个靶基因的表达,也可以通过几个miRNA的组合精细调控一个靶基因的表达。本实验室前期工作发现,在宫颈癌细胞中,miR-138靶定人端粒酶逆转录酶(human telomerase reverse transcriptase,hTERT)mRNA 3’UTR并负性调控其表达,抑制细胞的生长;miR-346靶定并上调hTERT的表达,促进细胞的生长,并且miR-138和mi R-346靶定hTERT mRNA 3’UTR的同一区域,二者竞争调控hTERT mRNA,维持其异常高表达水平。本课题在此基础上着重阐明miR-346结合hTERT mRNA 3’UTR上调其表达的分子机制。【方法】首先,宫颈癌细胞HeLa经放线菌素D(actinomycin D,Act D)处理之后用定量PCR(qRT-PCR)实验检测hTERT mRNA的表达水平,确定miR-346在转录水平还是转录后水平调控hTERT mRNA的表达。然后,用RNAhybrid软件预测miR-346/hTERT mRNA二聚体的可能二级结构,深入分析miR-346的模序;生物信息学预测miR-346的其它候选靶基因,并预测与这些候选靶基因形成二聚体后miR-346暴露的模序结构,分别选取与mi R-346杂交后暴露和不暴露类似miR-346/hTERT mRNA中miR-346模序的两个靶基因。利用荧光报告载体实验、qRT-PCR和western blot实验分别研究miR-346对hTERT的调控是否受AGO2的影响、模序突变型miR-346对hTERT表达调控的影响以及miR-346对ACVR2B表达调控的影响。在HeLa细胞中用AGO2的抗体进行RNA免疫沉淀实验(RNA IP,RIP),qRT-PCR检测AGO2复合体中miR-346和hTERT mRNA的富集水平。同时,利用MTT实验和平板克隆形成实验检测模序突变型miR-346对细胞活性和生长能力的影响。之后,将mi R-138/hTERT mRNA二级结构中暴露出的“UGAA”模序突变为野生型mir-346的“ccgcau”模序或突变型“aaaaua”模序,westernblot实验检测突变型mir-138对htert蛋白表达的影响。接下来,在hela细胞中单独改变grsf1或同时改变grsf1和野生型或模序突变型mir-346的表达水平,westernblot实验检测htert蛋白或acvr2b蛋白表达水平的改变,从而分析grsf1在mir-346上调htert和acvr2b表达过程中的作用。随后,利用rip实验和rna凝胶电泳迁移实验(electrophoreticmobilityshiftassay,emsa)实验验证grsf1与mir-346和/或htertmrna的相互作用。进一步地,用蔗糖密度梯度离心实验分离和纯化核糖体,qrt-pcr检测核糖体组分中mir-346和htertmrna的富集程度,分析mir-346是否募集htertmrna到核糖体以及该过程是否由grsf1介导。【结果】mir-346延长htertmrna的半衰期,增强htertmrna的稳定性。敲降ago2不影响mir-346对含有htert3’utr的绿色荧光蛋白报告基因和内源性htertmrna和蛋白的表达调控。ago2复合体中htertmrna的富集水平与mir-138(阳性对照)的表达水平呈正相关关系,与mir-346的表达水平呈负相关关系。rnahybrid软件预测mir-346/htertmrna二聚体的可能二级结构,二者杂交后mir-346暴露出“ccgcau”模序。突变mir-346的模序序列取消了野生型mir-346对报告基因和内源性htert表达的促进作用,解除了mir-346对细胞生长和增殖的促进作用。另外,含有mir-346“ccgcau”模序的mir-138促进htert蛋白的表达,而含有mir-346突变型“aaaaua”模序的mir-138抑制htert蛋白的表达。同时,生物信息学预测发现mir-346的候选靶基因有142个,rnahybrid软件分析mir-346与候选靶基因杂交的可能二级结构,最终选取了其中两个靶基因—activinareceptor,typeiib(acvr2b)和smadfamilymember3(smad3),其中,mir-346/acvr2bmrna二聚体暴露出mir-346的“ccgcau”模序,而mir-346/smad3mrna二聚体则不暴露类似的模序结构。mir-346靶定acvr2b3’utr并上调其的表达,同时,mir-346靶定smad33’utr并负性调控其表达。模序突变型mir-346解除了野生型mir-346对acvr2b的上调作用但不影响野生型mir-346对smad3的调控。htert蛋白和acvr2b蛋白的表达水平与grsf1水平呈正相关关系;敲降grsf1,mir-346上调htert和acvr2b表达的能力减弱,含有mir-346“ccgcau”模序的mir-138上调htert表达的能力减弱;突变mir-346的模序,grsf1促进htert和acvr2b表达的能力减弱。mir-346和hTERT mRNA在GRSF1复合体中富集,而且GRSF1复合体中hTERT mRNA的富集程度与miR-346的表达水平呈正相关关系。mi R-346和miR-346/hTERT mRNA3’UTR与GRSF1蛋白直接相互作用;miR-346模序突变后,该相互作用不存在。进一步分析发现,miR-346和hTERT mRNA在核糖体组分中共分布;而且,miR-346促进hTERT mRNA募集到核糖体,使其在核糖体组分中富集增加,分布曲线右移;突变miR-346的模序,hTERT mRNA在核糖体组分中富集减少,hTERT mRNA分布曲线左移。敲降GRSF1,miR-346介导的hTERT mRNA在核糖体组分中富集减少;模序突变型miR-346削弱了GRSF1介导的野生型mi R-346对hTERT mRNA到核糖体的募集。【结论】在宫颈癌细胞中,GRSF1以依赖miR-346“CCGCAU”模序的方式介导miR-346募集hTERT mRNA到核糖体并促进其翻译,该过程不依赖AGO2。
[Abstract]:[Objective] small RNA (microRNA, miRNA) is the most widely studied, the most explicit class of about 22nt, the endogenous small noncoding RNA molecule. Its classical mechanism is the mature miRNA assembly into RNA induced silencing complex (RNA-induced silencing complex, RISC), and the seed sequence is not translated according to the target gene mRNA. The degree of pairing of the region determines mRNA degradation or translation inhibition to regulate gene expression at post transcriptional levels. However, recent studies have found that miRNA can also increase gene expression at both transcriptional and post transcriptional levels, depending on the specific interaction between miRNA and target mRNA sequences, and with the miRNA ribonucleoprotein complex (microribonu). The specific components of cleoprotein, microRNP are closely related to the functional state of the cells. In addition, the regulatory network involved in miRNA is complex, which can regulate the expression of multiple target genes through a miRNA, and also fine regulate a target gene through several combinations of miRNA. MiR-138 target human telomerase reverse transcriptase (human telomerase reverse transcriptase, hTERT) mRNA 3 'UTR and negatively regulate its expression, inhibit cell growth, miR-346 target and up regulate the expression of hTERT, promote cell growth, and miR-138 and Mi targets determine the same area of 3', two competition regulation and regulation, To maintain its abnormal high expression level, this topic focuses on the molecular mechanism of miR-346 combined with hTERT mRNA 3 'UTR to increase its expression. [method] first, cervical cancer cells HeLa was treated with actinomycin D (actinomycin D, Act D) after the quantitative PCR (qRT-PCR) test was used to determine the expression level. The level or post transcriptional level regulates the expression of hTERT mRNA. Then, the RNAhybrid software is used to predict the possible two level structure of the miR-346/hTERT mRNA two polymer, and the sequence of miR-346 is deeply analyzed. Bioinformatics predicts the other candidate target genes of miR-346, and predicts the sequence structure of the miR-346 exposure after the formation of these candidate targets after the formation of the two polymer. Do not expose and expose the two target genes of miR-346 analogs in miR-346/hTERT mRNA after hybridization with MI R-346. Use the fluorescence report carrier experiment, qRT-PCR and Western blot experiments to study the effect of miR-346 on hTERT, and the effect of the mode sequence mutant miR-346 on the regulation of the expression. The effects of expression regulation. RNA immunoprecipitation test (RNA IP, RIP) in HeLa cells and qRT-PCR detection of miR-346 and hTERT mRNA enrichment in AGO2 complexes. Meanwhile, the effects of MTT experiments and flat clones on the activity and growth of the cells were detected by the MTT experiment and the flat clones. The "UGAA" model exposed in the T mRNA two structure is mutated to "ccgcau" or mutant "aaaaua" model of wild type mir-346, and the Westernblot test detects the effect of mutant miR-138 on the expression of hTERT protein. Expression level, Westernblot test detected the changes in the expression level of hTERT protein or acvr2b protein, and then analyzed the role of grsf1 in the mir-346 up regulation of hTERT and acvr2b expression. Subsequently, the experiments of rip and RNA gel electrophoresis (electrophoreticmobilityshiftassay, EMSA) experiments were used to verify the phase of grsf1 and / or the phase of acvr2b. Interaction. Further, the ribosomes were separated and purified with sucrose density gradient centrifugation, and the concentration of mir-346 and hTERTmRNA in the ribosome components was detected by qRT-PCR, and whether mir-346 raised hTERTmRNA to ribosomes and whether the process was mediated by grsf1. [results] mir-346 prolongs the half-life of hTERTmRNA and enhances the stability of hTERTmRNA. Knockdown ago2 does not affect mir-346's expression of htert3 'UTR green fluorescent protein reporter gene and endogenous hTERTmRNA and protein expression, the concentration of hTERTmRNA in the.Ago2 complex is positively correlated with the level of miR-138 (positive control), and is negatively related to the level of mir-346, and.Rnahybrid software predicts Mir. The possible two grade structure of -346/htertmrna two polymer, the two people exposed to the "ccgcau" motif after mir-346, and the sequence of mutant mir-346 abolished the promoting effect of wild type mir-346 on the expression of the reporter gene and endogenous hTERT, relieved the promoting effect of mir-346 on cell growth and proliferation. In addition, it contained mir-346 "ccgcau" motif. MiR-138 promotes the expression of hTERT protein, while miR-138 containing mir-346 mutant "aaaaua" model inhibits the expression of hTERT protein. At the same time, bioinformatics predicts that there are 142 candidate target genes for mir-346. Rnahybrid software analyses the possible two grade structure of the hybridization between mir-346 and candidate target genes, and finally selected two of the target genes - act. Ivinareceptor, typeiib (acvr2b) and smadfamilymember3 (Smad3), in which the mir-346/acvr2bmrna two polymer exposes the "ccgcau" motif of mir-346, while mir-346/smad3mrna two polymer does not expose a similar motile structure.Mir-346 targeting acvr2b3 'UTR and up-regulated its expression. Model sequence mutant mir-346 relieves the up-regulated effect of wild type mir-346 on acvr2b, but does not affect the regulation of Smad3 by wild type mir-346, and the expression level of.Htert protein and acvr2b protein is positively related to the level of grsf1, and the ability to knock down grsf1, mir-346 up hTERT and acvr2b expression is weakened. The ability of hTERT expression is weakened; the order of mutation of mir-346 and the ability of grsf1 to promote the expression of hTERT and acvr2b weaken the enrichment of.Mir-346 and hTERT mRNA in the GRSF1 complex, and the enrichment of hTERT mRNA in the GRSF1 complex is positively related to the level of the expression level. The interaction does not exist. Further analysis shows that the interaction does not exist. Further analysis shows that miR-346 and hTERT mRNA are distributed in the ribosome components; moreover, miR-346 promotes hTERT mRNA to raise ribosomes, enriching and increasing the ribosome in the ribosome components, and shifting the distribution curve to the right; the order of the mutated miR-346 and the enrichment and reduction of hTERT mRNA in the ribosome components. Less, hTERT mRNA distribution curve left shift. Knock down GRSF1, miR-346 mediated hTERT mRNA in ribosome components and decrease; mode sequence mutant miR-346 weakens GRSF1 mediated wild mi R-346 to the recruitment of hTERT mRNA to ribosome. Raising hTERT mRNA to the ribosome and promoting its translation does not depend on AGO2..

【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R737.33

【共引文献】