肝细胞癌中关键基因与microRNA的交互作用分析及EDNRB基因对肝癌细胞的作用研究

发布时间：2018-03-26 15:42

本文选题：肝细胞癌　切入点：微阵列分析　出处：《重庆医科大学》2017年硕士论文

【摘要】：第一部分肝细胞癌中关键基因与micro RNA的鉴别和交互作用的生物信息学分析目的:鉴别促进HCC发生发展的关键基因和miRNA及其潜在的分子机制。方法:从GEO数据库分别下载包含mRNA和miRNA表达谱的微阵列数据集GSE22058,GSE25097和GSE57958,并使用GEO2R进行癌及癌旁组织基因差异性分析。使用DAVID数据库对差异基因进行功能和通路富集分析。使用Cytoscape软件构建PPI网络。最后,使用miRDB预测差异表达的micro RNA的靶基因。结果:通过分析得到115个DEGs,其中包括52个上调基因和63个下调基因。功能和通路富集分析提示上调基因主要富集于染色体分离和细胞分裂过程,而下调基因主要参与补体激活、蛋白激活级联、羧酸代谢过程、含氧酸代谢过程及免疫反应。通过PPI网络分析筛选18个核心基因,其中ESR1与FOXO1、CXCL12和GNAO1具有密切的相互作用。同时,分析得到64个DEMs,其中hsa-mir-18a和hsa-mir-221等5种miRNA可能对ESR1有靶向调控作用。结论:ESR1及CXCL12等DEGs与has-mir-221等DEMs的相互作用可能在HCC的发生发展中起重要作用,这可能为HCC患者的诊断和治疗提供新的线索。第二部分EDNRB基因对肝癌细胞株SMMC-7721及Huh7的迁移和侵袭能力的影响目的:探讨EDNRB基因对人肝癌细胞迁移及侵袭能力的影响。方法:应用实时荧光定量PCR和Western blot法检测EDNRB基因mRNA和蛋白在人肝癌细胞株(SMMC-7721和Huh7),人肝细胞株LO2以及HCC组织及其癌旁的相应组织中的表达情况。通过慢病毒转染构建稳定过表达EDNRB基因的细胞株SMMC-7721-E和Huh7-E,应用CCK-8法及Transwell小室法分别检测EDNRB基因对肝癌细胞增殖、迁移及侵袭能力的影响。结果:EDNRB mRNA在SMMC-7721和Huh7细胞中低于LO2细胞;HCC组织中EDNRB mRNA的表达水平低于相匹配的癌旁组织(p0.01)。SMMC-7721和Huh7细胞中EDNRB蛋白表达低于LO2细胞;HCC组织中EDNRB蛋白的表达低于其相匹配的癌旁组织(p0.01)。EDNRB基因过表达对SMMC-7721和Huh7细胞的增殖无显著影响(p值均0.05),但能抑制其迁移和侵袭(p值均0.01)。结论:HCC中EDNRB基因的表达显著下调。EDNRB基因可抑制人肝癌细胞的迁移及侵袭。
[Abstract]:Part I: bioinformatics analysis of the identification and interaction between key genes and micro RNA in hepatocellular carcinoma objective: identification of key genes, miRNA and their potential molecular mechanisms promoting the development of HCC. Methods: from the GEO database. Do not download the microarray datasets GSE22058, GSE25097 and GSE57958, which contain mRNA and miRNA expression profiles, and use GEO2R to analyze the gene diversity of cancer and adjacent tissues. Use the DAVID database to analyze the function and pathway enrichment of differentially expressed genes. Construct the Cytoscape software. Build the PPI network. Finally, MiRDB was used to predict the target genes of differentially expressed micro RNA. Results: 115 DEGs were obtained, including 52 up-regulated genes and 63 down-regulated genes. Functional and pathway enrichment analysis showed that up-regulated genes were mainly enriched in chromosomes. Separation and cell division, The down-regulated genes are mainly involved in complement activation, protein activation cascade, carboxylic acid metabolism, oxygen-containing acid metabolism and immune response. Eighteen core genes were screened by PPI network analysis, in which ESR1 interacted closely with FOXO1CXCL12 and GNAO1. Among 64 demss, five kinds of miRNA, such as hsa-mir-18a and hsa-mir-221, may have targeted regulation on ESR1. Conclusion the interaction of DEGs such as CXCL12 and DEGs with has-mir-221 and DEMs may play an important role in the occurrence and development of HCC. This may provide a new clue for the diagnosis and treatment of HCC patients. Part two: the effect of EDNRB gene on the migration and invasion of hepatoma cell line SMMC-7721 and Huh7 objective: to explore the effect of EDNRB gene on the migration and invasion of human hepatoma cells. Methods: real-time fluorescence quantitative PCR and Western blot were used to detect the expression of EDNRB gene mRNA and protein in human hepatoma cell lines SMMC-7721 and Huh7, LO2, HCC and their adjacent tissues. Cell lines SMMC-7721-E and Huh7-E, which stably expressed EDNRB gene, were constructed by staining. The proliferation of hepatoma cells induced by EDNRB gene was detected by CCK-8 assay and Transwell chamber assay, respectively. Results the expression of EDNRB mRNA in SMMC-7721 and Huh7 cells was lower than that in LO2 cells, and the expression of EDNRB protein in Huh7 cells was lower than that in LO2 cells. The overexpression of white protein was lower than that of matched paracancerous tissues. The overexpression of EDNRB gene had no significant effect on the proliferation of SMMC-7721 and Huh7 cells, but could inhibit its migration and invasion. Conclusion the expression of EDNRB gene can be significantly down-regulated in SMMC-7721 and Huh7 cells. Conclusion\\\; It can inhibit the migration and invasion of human hepatoma cells.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.7

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