TGF-β1诱导子宫颈腺癌Hela细胞上皮间质转化mRNA和microRNA表达谱的研究

发布时间：2018-04-10 07:41

  本文选题：TGF-β1　切入点：子宫颈腺癌　出处：《西南医科大学》2017年硕士论文

【摘要】：目的:TGF-β1诱导子宫颈腺癌Hela细胞构建上皮间质转化模型,探讨EMT过程中mRNA和miRNA表达谱的变化规律。方法:1、10ng/ml TGF-β1作用于Hela细胞,构建宫颈腺癌细胞的EMT模型;并通过细胞形态学及RT-PCR鉴定EMT模型。2、应用基因芯片技术分别检测Hela细胞EMT模型组与对照组在mRNA及miRNA水平的表达,并通过生物信息学方法筛选出差异表达的mRNA及miRNA,分析mRNA和miRNA表达谱的变化。结果:1、TGF-β1刺激Hela细胞后逐渐出现间质细胞的特征,细胞由圆形或多边形逐渐变细长,伸出伪足,呈纺锤形或梭形,间隙变宽,由紧密逐渐变得疏松,细胞分布变得不规则。2、RT-PCR检测Hela细胞EMT模型E-cadherin、Vimentin的表达,结果显示,E-cadherin表达下调(t=-13.823,P=0.005),Vimentin表达上调(t=18.019,P=0.003),差异有统计学意义。3、mRNA芯片结果:差异表达的mRNA共计2235个,上调的差异mRNA有1116个,上调最显著的前三个基因为:CAPN10、PHACTR3、AMIGO2;下调的差异mRNA有1119个,下调最显著的前三个基因为:ECT2L、SOX14、CRTAC1。GO分析结果显示:上调mRNA参与的生物学过程有32个,包括RNA聚合酶II启动子的转录、细胞粘附、血管生成、细胞外基质组成、细胞增殖等;下调mrna未明显富集到明显相关的生物学过程(fdr0.05)。keggpassway分析提示:上调mrna被富集到的信号通路有14条,包括肿瘤信号通路、矿物质吸收、肥厚型心肌病、pi3k-akt、黏着斑等;下调mrna未富集到显著的信号通路(fdr0.05)。4、mirna芯片结果:差异表达的mirna共72个,其中上调的差异mirna有43个,上调最显著的前三个mirna为:hsa-mir-6805-5p、hsa-mir-4522、hsa-mir-6847-5p;下调的差异mirna有29个,下调最显著的前三个mirna为:hsa-mir-6860、hsa-mir-374b-5p、hsa-mir-4306。mirna靶基因预测结果:共筛选出2941个共同基因,其中上调和下调的mirna靶基因分别有1622个和1319个。go分析结果显示:上调mirna参与生物学过程有13个,包括神经系统发育、血管生成、表皮生长因子受体信号通路、神经营养因子trk受体信号通路、细胞黏附等。下调mirna参与生物学过程有11个,包括轴突导向、神经系统发育、同源细胞的粘附过程、正/负调控rnaⅡ启动子、血管内皮生长因子受体信号通路等。keggpassway分析提示:上调mirna被富集到的信号通路有39条,包括肿瘤通路、rap1信号通路、肿瘤内蛋白多糖、胞吞作用、轴突导向等;下调mirna被富集到的信号通路有15条,包括pi3k-akt、ras、ampk、camp、rap1等信号通路。结论:1、10ng/mltgf-β1能够构建宫颈腺癌hela细胞emt模型。2、mrna和mirna表达谱中差异表达的mrna和mirna参与多个生物学过程和信号通路,且miRNA可通过调控靶mRNA发挥作用。
[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，

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