本文選題：TGF-β1　切入點(diǎn)：子宮頸腺癌　出處：《西南醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:TGF-β1誘導(dǎo)子宮頸腺癌Hela細(xì)胞構(gòu)建上皮間質(zhì)轉(zhuǎn)化模型,探討EMT過程中mRNA和miRNA表達(dá)譜的變化規(guī)律。方法:1、10ng/ml TGF-β1作用于Hela細(xì)胞,構(gòu)建宮頸腺癌細(xì)胞的EMT模型;并通過細(xì)胞形態(tài)學(xué)及RT-PCR鑒定EMT模型。2、應(yīng)用基因芯片技術(shù)分別檢測(cè)Hela細(xì)胞EMT模型組與對(duì)照組在mRNA及miRNA水平的表達(dá),并通過生物信息學(xué)方法篩選出差異表達(dá)的mRNA及miRNA,分析mRNA和miRNA表達(dá)譜的變化。結(jié)果:1、TGF-β1刺激Hela細(xì)胞后逐漸出現(xiàn)間質(zhì)細(xì)胞的特征,細(xì)胞由圓形或多邊形逐漸變細(xì)長,伸出偽足,呈紡錘形或梭形,間隙變寬,由緊密逐漸變得疏松,細(xì)胞分布變得不規(guī)則。2、RT-PCR檢測(cè)Hela細(xì)胞EMT模型E-cadherin、Vimentin的表達(dá),結(jié)果顯示,E-cadherin表達(dá)下調(diào)(t=-13.823,P=0.005),Vimentin表達(dá)上調(diào)(t=18.019,P=0.003),差異有統(tǒng)計(jì)學(xué)意義。3、mRNA芯片結(jié)果:差異表達(dá)的mRNA共計(jì)2235個(gè),上調(diào)的差異mRNA有1116個(gè),上調(diào)最顯著的前三個(gè)基因?yàn)?CAPN10、PHACTR3、AMIGO2;下調(diào)的差異mRNA有1119個(gè),下調(diào)最顯著的前三個(gè)基因?yàn)?ECT2L、SOX14、CRTAC1。GO分析結(jié)果顯示:上調(diào)mRNA參與的生物學(xué)過程有32個(gè),包括RNA聚合酶II啟動(dòng)子的轉(zhuǎn)錄、細(xì)胞粘附、血管生成、細(xì)胞外基質(zhì)組成、細(xì)胞增殖等;下調(diào)mrna未明顯富集到明顯相關(guān)的生物學(xué)過程(fdr0.05)。keggpassway分析提示:上調(diào)mrna被富集到的信號(hào)通路有14條,包括腫瘤信號(hào)通路、礦物質(zhì)吸收、肥厚型心肌病、pi3k-akt、黏著斑等;下調(diào)mrna未富集到顯著的信號(hào)通路(fdr0.05)。4、mirna芯片結(jié)果:差異表達(dá)的mirna共72個(gè),其中上調(diào)的差異mirna有43個(gè),上調(diào)最顯著的前三個(gè)mirna為:hsa-mir-6805-5p、hsa-mir-4522、hsa-mir-6847-5p;下調(diào)的差異mirna有29個(gè),下調(diào)最顯著的前三個(gè)mirna為:hsa-mir-6860、hsa-mir-374b-5p、hsa-mir-4306。mirna靶基因預(yù)測(cè)結(jié)果:共篩選出2941個(gè)共同基因,其中上調(diào)和下調(diào)的mirna靶基因分別有1622個(gè)和1319個(gè)。go分析結(jié)果顯示:上調(diào)mirna參與生物學(xué)過程有13個(gè),包括神經(jīng)系統(tǒng)發(fā)育、血管生成、表皮生長因子受體信號(hào)通路、神經(jīng)營養(yǎng)因子trk受體信號(hào)通路、細(xì)胞黏附等。下調(diào)mirna參與生物學(xué)過程有11個(gè),包括軸突導(dǎo)向、神經(jīng)系統(tǒng)發(fā)育、同源細(xì)胞的粘附過程、正/負(fù)調(diào)控rnaⅡ啟動(dòng)子、血管內(nèi)皮生長因子受體信號(hào)通路等。keggpassway分析提示:上調(diào)mirna被富集到的信號(hào)通路有39條,包括腫瘤通路、rap1信號(hào)通路、腫瘤內(nèi)蛋白多糖、胞吞作用、軸突導(dǎo)向等;下調(diào)mirna被富集到的信號(hào)通路有15條,包括pi3k-akt、ras、ampk、camp、rap1等信號(hào)通路。結(jié)論:1、10ng/mltgf-β1能夠構(gòu)建宮頸腺癌hela細(xì)胞emt模型。2、mrna和mirna表達(dá)譜中差異表達(dá)的mrna和mirna參與多個(gè)生物學(xué)過程和信號(hào)通路,且miRNA可通過調(diào)控靶mRNA發(fā)揮作用。
[Abstract]:Objective to construct the epithelial interstitial transformation model of Hela cells induced by TGF- 尾 1, and to investigate the changes of mRNA and miRNA expression profiles in the process of EMT.Methods 10 ng / ml TGF- 尾 1 of 10 ng / ml TGF- 尾 1 was used to construct the EMT model of cervical adenocarcinoma cells, and the EMT model was identified by cell morphology and RT-PCR. The expression of mRNA and miRNA in Hela cell EMT model group and control group were detected by gene chip technique.The differentially expressed mRNA and miRNAs were screened by bioinformatics, and the changes of mRNA and miRNA expression profiles were analyzed.Results the interstitial cells appeared in the Hela cells stimulated by TGF- 尾 1. The cells gradually became thin and elongated from a circular or polygon to a spindle-shaped or fusiform. The gap became wider and loosened gradually.The expression of E-cadherin in EMT model of Hela cells was detected by RT-PCR. The results showed that the expression of E-cadherin was down-regulated, and the expression of Vimentin was up-regulated.The first three up-regulated genes were: 1 / CAPN10 / PHACTR3AmiGO2; the down-regulated mRNA was 1119, and the first three down-regulated genes were: 1. 0% ECT2LX SOX14 CRTAC1.GO results showed that there were 32 biological processes involved in up-regulation of mRNA, including transcription of RNA polymerase II promoter and cell adhesion.Angiogenesis, extracellular matrix composition, cell proliferation, etc. Down-regulation of mrna was not significantly enriched to the related biological process. Keg passway analysis showed that there were 14 signaling pathways that up-regulated mrna enrichment, including tumor signaling pathway and mineral absorption.Including nervous system development, angiogenesis, epidermal growth factor receptor signaling pathway, neurotrophic factor trk receptor signal pathway, cell adhesion and so on.Down-regulation of mirna is involved in 11 biological processes, including axon orientation, nervous system development, homologous cell adhesion, and positive / negative regulation of rna 鈪，

本文編號(hào)：1730297