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GRSF1介導miR-346上調(diào)hTERT表達的作用與機制研究

發(fā)布時間:2018-04-30 04:12

  本文選題:microRNA + mi; 參考:《天津醫(yī)科大學》2015年博士論文


【摘要】:【目的】微小RNA(microRNA,miRNA)是近十幾年來研究最廣泛、最明確的一類大小約22nt、內(nèi)源性的小非編碼RNA分子。其經(jīng)典的作用機制是成熟的miRNA組裝進入RNA誘導的沉默復合體(RNA-induced silencing complex,RISC),種子序列依據(jù)與靶基因mRNA非翻譯區(qū)的配對程度決定mRNA降解還是翻譯抑制,從而在轉錄后水平調(diào)節(jié)基因的表達。但是,最近的研究發(fā)現(xiàn),miRNA還可以在轉錄和轉錄后水平上調(diào)基因的表達,這依賴于miRNA與靶mRNA序列的特異性相互作用,并且與miRNA核糖核蛋白復合體(microribonucleoprotein,microRNP)的具體成分及細胞的功能狀態(tài)密切相關。另外,miRNA參與的調(diào)控網(wǎng)絡是復雜的,既可以通過一個miRNA調(diào)控多個靶基因的表達,也可以通過幾個miRNA的組合精細調(diào)控一個靶基因的表達。本實驗室前期工作發(fā)現(xiàn),在宮頸癌細胞中,miR-138靶定人端粒酶逆轉錄酶(human telomerase reverse transcriptase,hTERT)mRNA 3’UTR并負性調(diào)控其表達,抑制細胞的生長;miR-346靶定并上調(diào)hTERT的表達,促進細胞的生長,并且miR-138和mi R-346靶定hTERT mRNA 3’UTR的同一區(qū)域,二者競爭調(diào)控hTERT mRNA,維持其異常高表達水平。本課題在此基礎上著重闡明miR-346結合hTERT mRNA 3’UTR上調(diào)其表達的分子機制。【方法】首先,宮頸癌細胞HeLa經(jīng)放線菌素D(actinomycin D,Act D)處理之后用定量PCR(qRT-PCR)實驗檢測hTERT mRNA的表達水平,確定miR-346在轉錄水平還是轉錄后水平調(diào)控hTERT mRNA的表達。然后,用RNAhybrid軟件預測miR-346/hTERT mRNA二聚體的可能二級結構,深入分析miR-346的模序;生物信息學預測miR-346的其它候選靶基因,并預測與這些候選靶基因形成二聚體后miR-346暴露的模序結構,分別選取與mi R-346雜交后暴露和不暴露類似miR-346/hTERT mRNA中miR-346模序的兩個靶基因。利用熒光報告載體實驗、qRT-PCR和western blot實驗分別研究miR-346對hTERT的調(diào)控是否受AGO2的影響、模序突變型miR-346對hTERT表達調(diào)控的影響以及miR-346對ACVR2B表達調(diào)控的影響。在HeLa細胞中用AGO2的抗體進行RNA免疫沉淀實驗(RNA IP,RIP),qRT-PCR檢測AGO2復合體中miR-346和hTERT mRNA的富集水平。同時,利用MTT實驗和平板克隆形成實驗檢測模序突變型miR-346對細胞活性和生長能力的影響。之后,將mi R-138/hTERT mRNA二級結構中暴露出的“UGAA”模序突變?yōu)橐吧蚼ir-346的“ccgcau”模序或突變型“aaaaua”模序,westernblot實驗檢測突變型mir-138對htert蛋白表達的影響。接下來,在hela細胞中單獨改變grsf1或同時改變grsf1和野生型或模序突變型mir-346的表達水平,westernblot實驗檢測htert蛋白或acvr2b蛋白表達水平的改變,從而分析grsf1在mir-346上調(diào)htert和acvr2b表達過程中的作用。隨后,利用rip實驗和rna凝膠電泳遷移實驗(electrophoreticmobilityshiftassay,emsa)實驗驗證grsf1與mir-346和/或htertmrna的相互作用。進一步地,用蔗糖密度梯度離心實驗分離和純化核糖體,qrt-pcr檢測核糖體組分中mir-346和htertmrna的富集程度,分析mir-346是否募集htertmrna到核糖體以及該過程是否由grsf1介導!窘Y果】mir-346延長htertmrna的半衰期,增強htertmrna的穩(wěn)定性。敲降ago2不影響mir-346對含有htert3’utr的綠色熒光蛋白報告基因和內(nèi)源性htertmrna和蛋白的表達調(diào)控。ago2復合體中htertmrna的富集水平與mir-138(陽性對照)的表達水平呈正相關關系,與mir-346的表達水平呈負相關關系。rnahybrid軟件預測mir-346/htertmrna二聚體的可能二級結構,二者雜交后mir-346暴露出“ccgcau”模序。突變mir-346的模序序列取消了野生型mir-346對報告基因和內(nèi)源性htert表達的促進作用,解除了mir-346對細胞生長和增殖的促進作用。另外,含有mir-346“ccgcau”模序的mir-138促進htert蛋白的表達,而含有mir-346突變型“aaaaua”模序的mir-138抑制htert蛋白的表達。同時,生物信息學預測發(fā)現(xiàn)mir-346的候選靶基因有142個,rnahybrid軟件分析mir-346與候選靶基因雜交的可能二級結構,最終選取了其中兩個靶基因—activinareceptor,typeiib(acvr2b)和smadfamilymember3(smad3),其中,mir-346/acvr2bmrna二聚體暴露出mir-346的“ccgcau”模序,而mir-346/smad3mrna二聚體則不暴露類似的模序結構。mir-346靶定acvr2b3’utr并上調(diào)其的表達,同時,mir-346靶定smad33’utr并負性調(diào)控其表達。模序突變型mir-346解除了野生型mir-346對acvr2b的上調(diào)作用但不影響野生型mir-346對smad3的調(diào)控。htert蛋白和acvr2b蛋白的表達水平與grsf1水平呈正相關關系;敲降grsf1,mir-346上調(diào)htert和acvr2b表達的能力減弱,含有mir-346“ccgcau”模序的mir-138上調(diào)htert表達的能力減弱;突變mir-346的模序,grsf1促進htert和acvr2b表達的能力減弱。mir-346和hTERT mRNA在GRSF1復合體中富集,而且GRSF1復合體中hTERT mRNA的富集程度與miR-346的表達水平呈正相關關系。mi R-346和miR-346/hTERT mRNA3’UTR與GRSF1蛋白直接相互作用;miR-346模序突變后,該相互作用不存在。進一步分析發(fā)現(xiàn),miR-346和hTERT mRNA在核糖體組分中共分布;而且,miR-346促進hTERT mRNA募集到核糖體,使其在核糖體組分中富集增加,分布曲線右移;突變miR-346的模序,hTERT mRNA在核糖體組分中富集減少,hTERT mRNA分布曲線左移。敲降GRSF1,miR-346介導的hTERT mRNA在核糖體組分中富集減少;模序突變型miR-346削弱了GRSF1介導的野生型mi R-346對hTERT mRNA到核糖體的募集!窘Y論】在宮頸癌細胞中,GRSF1以依賴miR-346“CCGCAU”模序的方式介導miR-346募集hTERT mRNA到核糖體并促進其翻譯,該過程不依賴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..

【學位授予單位】:天津醫(yī)科大學
【學位級別】:博士
【學位授予年份】:2015
【分類號】:R737.33

【共引文獻】

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