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

【摘要】：【目的】微小RNA(microRNA,miRNA)是近十幾年來(lái)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(jī)制是成熟的miRNA組裝進(jìn)入RNA誘導(dǎo)的沉默復(fù)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(duì)程度決定mRNA降解還是翻譯抑制,從而在轉(zhuǎn)錄后水平調(diào)節(jié)基因的表達(dá)。但是,最近的研究發(fā)現(xiàn),miRNA還可以在轉(zhuǎn)錄和轉(zhuǎn)錄后水平上調(diào)基因的表達(dá),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(fù)合體(microribonucleoprotein,microRNP)的具體成分及細(xì)胞的功能狀態(tài)密切相關(guān)。另外,miRNA參與的調(diào)控網(wǎng)絡(luò)是復(fù)雜的,既可以通過一個(gè)miRNA調(diào)控多個(gè)靶基因的表達(dá),也可以通過幾個(gè)miRNA的組合精細(xì)調(diào)控一個(gè)靶基因的表達(dá)。本實(shí)驗(yàn)室前期工作發(fā)現(xiàn),在宮頸癌細(xì)胞中,miR-138靶定人端粒酶逆轉(zhuǎn)錄酶(human telomerase reverse transcriptase,hTERT)mRNA 3’UTR并負(fù)性調(diào)控其表達(dá),抑制細(xì)胞的生長(zhǎng);miR-346靶定并上調(diào)hTERT的表達(dá),促進(jìn)細(xì)胞的生長(zhǎng),并且miR-138和mi R-346靶定hTERT mRNA 3’UTR的同一區(qū)域,二者競(jìng)爭(zhēng)調(diào)控hTERT mRNA,維持其異常高表達(dá)水平。本課題在此基礎(chǔ)上著重闡明miR-346結(jié)合hTERT mRNA 3’UTR上調(diào)其表達(dá)的分子機(jī)制。【方法】首先,宮頸癌細(xì)胞HeLa經(jīng)放線菌素D(actinomycin D,Act D)處理之后用定量PCR(qRT-PCR)實(shí)驗(yàn)檢測(cè)hTERT mRNA的表達(dá)水平,確定miR-346在轉(zhuǎn)錄水平還是轉(zhuǎn)錄后水平調(diào)控hTERT mRNA的表達(dá)。然后,用RNAhybrid軟件預(yù)測(cè)miR-346/hTERT mRNA二聚體的可能二級(jí)結(jié)構(gòu),深入分析miR-346的模序;生物信息學(xué)預(yù)測(cè)miR-346的其它候選靶基因,并預(yù)測(cè)與這些候選靶基因形成二聚體后miR-346暴露的模序結(jié)構(gòu),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(gè)靶基因。利用熒光報(bào)告載體實(shí)驗(yàn)、qRT-PCR和western blot實(shí)驗(yàn)分別研究miR-346對(duì)hTERT的調(diào)控是否受AGO2的影響、模序突變型miR-346對(duì)hTERT表達(dá)調(diào)控的影響以及miR-346對(duì)ACVR2B表達(dá)調(diào)控的影響。在HeLa細(xì)胞中用AGO2的抗體進(jìn)行RNA免疫沉淀實(shí)驗(yàn)(RNA IP,RIP),qRT-PCR檢測(cè)AGO2復(fù)合體中miR-346和hTERT mRNA的富集水平。同時(shí),利用MTT實(shí)驗(yàn)和平板克隆形成實(shí)驗(yàn)檢測(cè)模序突變型miR-346對(duì)細(xì)胞活性和生長(zhǎng)能力的影響。之后,將mi R-138/hTERT mRNA二級(jí)結(jié)構(gòu)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(shí)驗(yàn)檢測(cè)突變型mir-138對(duì)htert蛋白表達(dá)的影響。接下來(lái),在hela細(xì)胞中單獨(dú)改變grsf1或同時(shí)改變grsf1和野生型或模序突變型mir-346的表達(dá)水平,westernblot實(shí)驗(yàn)檢測(cè)htert蛋白或acvr2b蛋白表達(dá)水平的改變,從而分析grsf1在mir-346上調(diào)htert和acvr2b表達(dá)過程中的作用。隨后,利用rip實(shí)驗(yàn)和rna凝膠電泳遷移實(shí)驗(yàn)(electrophoreticmobilityshiftassay,emsa)實(shí)驗(yàn)驗(yàn)證grsf1與mir-346和/或htertmrna的相互作用。進(jìn)一步地,用蔗糖密度梯度離心實(shí)驗(yàn)分離和純化核糖體,qrt-pcr檢測(cè)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(dǎo)�！窘Y(jié)果】mir-346延長(zhǎng)htertmrna的半衰期,增強(qiáng)htertmrna的穩(wěn)定性。敲降ago2不影響mir-346對(duì)含有htert3’utr的綠色熒光蛋白報(bào)告基因和內(nèi)源性htertmrna和蛋白的表達(dá)調(diào)控。ago2復(fù)合體中htertmrna的富集水平與mir-138(陽(yáng)性對(duì)照)的表達(dá)水平呈正相關(guān)關(guān)系,與mir-346的表達(dá)水平呈負(fù)相關(guān)關(guān)系。rnahybrid軟件預(yù)測(cè)mir-346/htertmrna二聚體的可能二級(jí)結(jié)構(gòu),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(duì)報(bào)告基因和內(nèi)源性htert表達(dá)的促進(jìn)作用,解除了mir-346對(duì)細(xì)胞生長(zhǎng)和增殖的促進(jìn)作用。另外,含有mir-346“ccgcau”模序的mir-138促進(jìn)htert蛋白的表達(dá),而含有mir-346突變型“aaaaua”模序的mir-138抑制htert蛋白的表達(dá)。同時(shí),生物信息學(xué)預(yù)測(cè)發(fā)現(xiàn)mir-346的候選靶基因有142個(gè),rnahybrid軟件分析mir-346與候選靶基因雜交的可能二級(jí)結(jié)構(gòu),最終選取了其中兩個(gè)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(jié)構(gòu)。mir-346靶定acvr2b3’utr并上調(diào)其的表達(dá),同時(shí),mir-346靶定smad33’utr并負(fù)性調(diào)控其表達(dá)。模序突變型mir-346解除了野生型mir-346對(duì)acvr2b的上調(diào)作用但不影響野生型mir-346對(duì)smad3的調(diào)控。htert蛋白和acvr2b蛋白的表達(dá)水平與grsf1水平呈正相關(guān)關(guān)系;敲降grsf1,mir-346上調(diào)htert和acvr2b表達(dá)的能力減弱,含有mir-346“ccgcau”模序的mir-138上調(diào)htert表達(dá)的能力減弱;突變mir-346的模序,grsf1促進(jìn)htert和acvr2b表達(dá)的能力減弱。mir-346和hTERT mRNA在GRSF1復(fù)合體中富集,而且GRSF1復(fù)合體中hTERT mRNA的富集程度與miR-346的表達(dá)水平呈正相關(guān)關(guān)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(jìn)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(jìn)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(dǎo)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(dǎo)的野生型mi R-346對(duì)hTERT mRNA到核糖體的募集�！窘Y(jié)論】在宮頸癌細(xì)胞中,GRSF1以依賴miR-346“CCGCAU”模序的方式介導(dǎo)miR-346募集hTERT mRNA到核糖體并促進(jìn)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(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R737.33

【共引文獻(xiàn)】

相關(guān)期刊論文 前10條

1 袁泉;柳滿然;曾宗躍;周旭春;;microRNA與胃癌耐藥的關(guān)系[J];國(guó)際檢驗(yàn)醫(yī)學(xué)雜志;2013年17期

2 Wenwen Jia;Wen Chen;Jiuhong Kang;;The Functions of MicroRNAs and Long Non-coding RNAs in Embryonic and Induced Pluripotent Stem Cells[J];Genomics,Proteomics & Bioinformatics;2013年05期

3 宋菲;王欣;錢平;唐順明;趙巧玲;郭錫杰;沈興家;;家蠶Bmo-miR-2739對(duì)絲素重鏈基因Fib-H表達(dá)的調(diào)控作用[J];蠶業(yè)科學(xué);2014年03期

4 焦良波;陳濤;胡衛(wèi);;肝癌干細(xì)胞中microRNAs的功能研究進(jìn)展[J];廣東醫(yī)學(xué);2014年18期

5 張百紅;岳紅云;;腫瘤的圖像特征[J];醫(yī)學(xué)爭(zhēng)鳴;2014年05期

6 Jeff P.Bruce;Fei-Fei Liu;;MicroRNAs in nasopharyngeal carcinoma[J];Chinese Journal of Cancer;2014年11期

7 宋菲;王欣;錢平;陳晨;范洋洋;沈興家;;microRNA的功能及家蠶microRNA的研究進(jìn)展[J];蠶業(yè)科學(xué);2015年01期

8 張慶;趙博欣;王勝奇;梁倩瑩;楊富恒;王雅甜;蔡云;李國(guó)鋒;;原花青素通過調(diào)節(jié)微小RNA-27a與微小RNA-451抑制多藥耐藥基因1 mRNA的表達(dá)[J];國(guó)際藥學(xué)研究雜志;2015年04期

9 范洋洋;陳晨;王欣;宋菲;唐順明;易詠竹;沈興家;;Bmo-miR-2755*對(duì)絲素蛋白基因BmFib-L和BmP25表達(dá)的調(diào)控作用[J];蠶業(yè)科學(xué);2015年05期

10 FENG XueZhu;GUANG ShouHong;;Non-coding RNAs mediate the rearrangements of genomic DNA in ciliates[J];Science China(Life Sciences);2013年10期

相關(guān)博士學(xué)位論文 前10條

1 陳琦;慢病毒介導(dǎo)的FoxP2 knockdown蝙蝠模型建立與功能研究及長(zhǎng)翼蝠小RNA轉(zhuǎn)錄組的分析研究[D];華東師范大學(xué);2013年

2 施建東;甲型肝炎病毒編碼microRNAs分子的預(yù)測(cè)、鑒定及功能研究[D];北京協(xié)和醫(yī)學(xué)院;2013年

3 陳弘;小RNA誘導(dǎo)INTS6基因激活對(duì)去勢(shì)抵抗性前列腺癌的作用及機(jī)制研究[D];浙江大學(xué);2013年

4 王晨;葡萄microRNA及其靶基因的識(shí)別、鑒定及作用方式研究[D];南京農(nóng)業(yè)大學(xué);2012年

5 劉佳;小鼠肝細(xì)胞AMPK相關(guān)microRNAs的篩選及功能研究[D];天津醫(yī)科大學(xué);2013年

6 韋薇;Argonaute2蛋白和DNA雙鏈斷裂誘導(dǎo)產(chǎn)生的小RNA調(diào)控同源重組的機(jī)制研究[D];北京協(xié)和醫(yī)學(xué)院;2013年

7 黃添枝;RNA干擾在對(duì)蝦抗病毒免疫中的作用[D];浙江大學(xué);2013年

8 張世芳;綿羊miRNA轉(zhuǎn)錄組的鑒定與特征分析[D];中國(guó)農(nóng)業(yè)科學(xué)院;2013年

9 吳淡森;p15-piRNAs介導(dǎo)白血病細(xì)胞p15~(INK4b)基因DNA與組蛋白甲基化修飾機(jī)制研究[D];福建醫(yī)科大學(xué);2013年

10 龔靜;人類microRNA基因DNA和RNA水平變異系統(tǒng)分析[D];華中科技大學(xué);2013年

相關(guān)碩士學(xué)位論文 前10條

1 魯明;miR-206對(duì)牛骨骼肌衛(wèi)星細(xì)胞分化的影響研究[D];東北農(nóng)業(yè)大學(xué);2013年

2 周麗;miR-135a/b在肺癌細(xì)胞耐藥中的作用及相關(guān)機(jī)制研究[D];南京醫(yī)科大學(xué);2013年

3 張小龍;miR-125b在乳腺癌中對(duì)MKNK2的靶向調(diào)控[D];華東師范大學(xué);2013年

4 張?jiān)诒?糖尿病大鼠心肌梗死后缺血區(qū)miRNA-503及其靶基因CCNE1和cdc25A的動(dòng)態(tài)變化[D];福建醫(yī)科大學(xué);2013年

5 趙娟霞;靶向作用喉癌候選抑瘤基因LCRG1的microRNAs的初步篩選研究[D];南華大學(xué);2013年

6 李艷艷;MiR-146b-5p異常減少在膠質(zhì)瘤發(fā)生發(fā)展中的作用及其機(jī)制研究[D];天津醫(yī)科大學(xué);2013年

7 吳紅明;小鼠酒精性心肌病相關(guān)microRNA表達(dá)譜分析[D];昆明醫(yī)科大學(xué);2013年

8 卞爾保;DNA甲基化在大鼠肝纖維化中的作用及其部分機(jī)制研究[D];安徽醫(yī)科大學(xué);2013年

9 陳五軍;MiR-27a/b靶向沉默ABCA1對(duì)巨噬細(xì)胞膽固醇流出的影響[D];南華大學(xué);2013年

10 葉建蔚;小鼠肝泡型包蟲病中miRNAs表達(dá)譜的初步篩選及miR-106b-25基因簇的表達(dá)和意義[D];新疆醫(yī)科大學(xué);2013年

，

本文編號(hào)：1823011