沉默肝癌细胞中NET-1基因后的抑癌作用及相关分子机制的研究

发布时间：2018-04-24 03:41

本文选题：肝细胞性肝癌 + NET-1　；参考：《南通大学》2016年硕士论文

【摘要】：目的在肝细胞肝癌(hepatocellular carcinoma,HCC)中研究短发夹RNA(short hairpin RNA,shRNA)沉默NET-1(TSPAN1)基因后的抑癌作用及其相关的分子机制。方法1.实时定量PCR(Real-time quantitative polymerase chain reaction,RT-q PCR)和蛋白免疫印迹(Western Blots,WB)检测不同HCC细胞株(MHCC97H、MHCC97L、HepG2、SMMC7721)中NET-1基因mRNA和蛋白的表达,筛选本研究的HCC靶细胞。2.构建靶向NET-1的全位点核酸干扰(RNAi)文库,筛选沉默NET-1的最佳靶点,进而构建靶向NET-1的shRNA(NET-1 shRNA)转染靶细胞。MTT、流式细胞仪检测、Hoechst细胞凋亡检测和Annexin V/PI染色流式细胞仪检测NET-1 shRNA对HCC细胞增殖与凋亡的干预作用。3.通过cDNA基因芯片检测NET-1 shRNA处理HCC细胞后基因表达差异,通过GO和KEGG富集分析探索差异基因的生物学功能及相关的信号通路。RT-qPCR、WB、免疫共沉淀(Co-Immunoprecipitation,Co-IP)和免疫荧光(Immunofluorescencetechnique,IF)共定位方法分别检测靶细胞内NET-1、MDM4、CHEK2、P53表达的相关性;并分别构建NET-1、MDM4、P53过表达载体体外转染HEK293T细胞,Co-IP方法外源性检测NET-1、MDM4、P53细胞内环境中表达的相关性,探究NET-1 shRNA激活P53抑制HCC的分子机制。结果1.不同hcc细胞株(mhcc97h、mhcc97l、hepg2、smmc7721)中net-1mrna和蛋白的表达水平均显著高于正常肝细胞(lo2)(p0.05)。在mhcc97h和mhcc97l细胞中的表达水平显著高于hepg2、smmc7721细胞(p0.05),故选用高转移潜能hcc细胞株(mhcc97h)和低转移潜能hcc细胞株(mhcc97l)作为本文研究的靶细胞。2.成功构建覆盖net-1基因的rnai全位点文库(143个克隆),在选取沉默效率较高的5个位点中进一步筛选并得到最优沉默靶点,构建net-1shrna。3.net-1蛋白定位于靶细胞胞质或胞膜。与阴性对照shrnanc处理细胞相比,net-1shrna处理靶细胞后显著下调了net-1mrna和蛋白表达水平,细胞被显著阻滞在g1期,增殖能力下降,而细胞凋亡率显著增加(均p0.05)。4.affymetrix基因芯片筛选出net-1shrna处理靶细胞后基因mrna表达水平呈现两倍以上变化的差异基因。在mhcc97h细胞中有7393个,其中3389个基因表达上调,4004个基因表达下调;在mhcc97l细胞中有115个,其中55个基因表达上调,60个基因表达下调。5.通过go和kegg富集分析net-1shrna处理后差异基因的主要生物学功能和相关的信号通路。在mhcc97h细胞中差异基因参与的生物学功能主要有:细胞间的信号传导、细胞粘附、细胞凋亡、细胞周期、细胞增殖调控、血管生成等;参与的信号通路主要有:代谢途径、癌症通路、p53信号通路、丝裂原活化蛋白激酶信号通路、tgf-β信号通路、vegf信号通路、mtor信号通路等。在mhcc97l细胞中差异基因参与的生物学功能主要有:基因转录调控、细胞增殖、生长调节、蛋白磷酸化调控、蛋白核转录的调控、细胞周期阻滞调控等;参与的信号通路主要有:癌症通路、粘着连接、jak-stat信号通路、notch信号通路、rig-i样受体信号通路等。6.通过rt-qpcr、蛋白免疫印迹和免疫共定位等方法验证基因芯片检测的差异表达基因。与shrnanc处理相比,net-1shrna处理mhcc97h细胞中chek2mrna和蛋白表达量显著增加(p0.05),虽然net-1和chek2的相互作用尚不明确,但chek2和p53蛋白在癌细胞中存在胞核内共定位和蛋白相互作用。在net-1shrna处理mhcc97l细胞中mdm4mrna和蛋白表达量显著减少,该蛋白表达定位于胞质和胞核。通过内源性免疫共沉淀方法检测发现net-1与mdm4,mdm4与p53在hcc细胞中存在相互作用,且在HEK293T细胞中通过外源性检测得到进一步验证。结论1.构建覆盖NET-1基因全长的全位点RNAi文库,并筛选得到高效沉默NET-1靶基因的RNAi靶点序列。2.本文设计与构建的NET-1 shRNA转染HCC细胞后能显著下调细胞中NET-1的表达,抑制癌细胞增殖、促进癌细胞凋亡,显示较好的抑癌作用。3.NET-1 shRNA转染HCC细胞后可引起相关基因表达的显著变化,其主要差异表达基因的生物学功能和所涉及到的信号通路均与细胞增殖、凋亡的基因调控有关。4.NET-1 shRNA下调癌细胞中NET-1基因后的抑癌作用分子机制在不同转移潜能的HCC细胞中存在差异。分别通过上调高转移潜能癌细胞中CHEK2表达,或下调低转移潜能癌细胞中MDM4表达,殊途同归最终激活了抑癌基因P53的机制来实现抑制增殖和促进凋亡的作用。5.NET-1是干预HCC的有效靶点。本研究为NET-1 shRNA进一步的开发,用于临床治疗提供了理论和实验依据。
[Abstract]:Objective to study the tumor suppressor effect and its molecular mechanism after the silent NET-1 (TSPAN1) gene of the short hairpin RNA (short hairpin RNA, shRNA) in hepatocellular carcinoma (HCC). Method 1. real-time quantitative PCR (Real-time) and protein immunoblotting The expression of mRNA and protein of NET-1 gene in different HCC cell lines (MHCC97H, MHCC97L, HepG2, SMMC7721), screening the HCC target cells in this study to construct the whole site nucleic acid interference (RNAi) library of target NET-1, screening the best target of the silent NET-1. Detection of T cell apoptosis and Annexin V/PI staining flow cytometry the interference of NET-1 shRNA on the proliferation and apoptosis of HCC cells.3. through the cDNA gene chip to detect the difference of gene expression after NET-1 shRNA processing HCC cells, and to explore the biological power and related signal pathways of the differential genes through GO and enrichment analysis. Co-Immunoprecipitation (Co-IP) and immunofluorescence (Immunofluorescencetechnique, IF) Co location method were used to detect the correlation of NET-1, MDM4, CHEK2, P53 expression in the target cells, and NET-1, MDM4, P53 overexpressed vectors were transfected into the cells in vitro. To explore the molecular mechanism of NET-1 shRNA activation of P53 to inhibit HCC. Results 1. the expression level of net-1mrna and protein in different HCC cell lines (MHCC97H, mhcc97l, HepG2, SMMC7721) was significantly higher than that of normal liver cells (LO2). The metastatic potential HCC cell line (MHCC97H) and the low metastatic potential HCC cell line (mhcc97l), as the target cell of this paper, have successfully constructed the RNAi full site library (143 clones) covering NET-1 gene, and selected the best silent target in the 5 loci with high silencing efficiency, and constructed the net-1shrna.3.net-1 protein to target the target. Cell cytoplasm or cell membrane. Compared with negative control shrnanc treated cells, net-1shrna significantly lowered the level of net-1mrna and protein expression after the target cell treatment. The cell was significantly blocked in the G1 phase and the proliferation ability decreased, while the apoptosis rate increased significantly (P0.05).4.affymetrix based gene chip screened the gene mRNA after the net-1shrna treatment of the target cells. There were 7393 differentially expressed genes with two times more than two times, of which 3389 genes were up-regulated, 4004 genes were downregulated, 115 in mhcc97l cells, 55 of which were up-regulated, and 60 genes were down regulated by go and KEGG rich set analysis of the major genes of differential genes after net-1shrna treatment. The biological functions of differential genes involved in MHCC97H cells are mainly signal transduction, cell adhesion, cell apoptosis, cell cycle, cell proliferation regulation, angiogenesis, and so on. The signal pathways involved are mainly metabolite pathway, cancer pathway, p53 signaling pathway, mitogen activated protein kinase Signal pathways, tgf- beta signaling pathways, VEGF signaling pathways, and mTOR signaling pathways. The biological functions of differentially genes involved in mhcc97l cells include gene transcription regulation, cell proliferation, growth regulation, protein phosphorylation regulation, regulation of protein nucleus transcription, and cell cycle arrest regulation, and the main signaling pathways involved are cancer pathways, Adhesion, JAK-STAT signaling pathway, Notch signaling pathway, RIG-I like receptor signaling pathway and other.6. through RT-qPCR, protein immunoblotting and immunoblotting were used to verify the differentially expressed genes of gene chip detection. Compared with shrnanc treatment, chek2mrna and protein expression in net-1shrna treated MHCC97H cells increased significantly (P0.05), although ne The interaction of T-1 and CHEK2 is not clear, but the co localization and protein interaction of CHEK2 and p53 proteins exist in the cell nuclei of the cancer cells. The expression of mdm4mrna and protein in the net-1shrna treatment mhcc97l cells is significantly reduced, and the protein expression is located in the cytoplasm and nucleus. NET-1 and MDM4, MDM4 are detected by endogenous immunoprecipitation method. Interaction with p53 in HCC cells and further verification by exogenous detection in HEK293T cells. Conclusion 1. construct a full site RNAi library covering the full length of NET-1 gene, and screen the RNAi target sequence of the target gene for highly silenced NET-1, which is designed and constructed by the NET-1 shRNA transfected to HCC cells. The expression of NET-1 in the cell can inhibit the proliferation of cancer cells and promote the apoptosis of cancer cells. It shows that the good tumor suppressor effect of.3.NET-1 shRNA transfection to HCC cells can cause significant changes in the expression of related genes. The biological functions of the main differentially expressed genes and the signal pathways involved are related to the proliferation of cells and the regulation of.4.NET-1 sh on the gene regulation of apoptosis. The molecular mechanism of the inhibitory effect of RNA on the NET-1 gene in cancer cells is different in the HCC cells with different metastatic potential. By up regulating the expression of CHEK2 in the cancer cells with high metastatic potential or decreasing the expression of MDM4 in the cancer cells with low metastatic potential, the mechanism of the tumor suppressor based on P53 is ultimately activated to inhibit proliferation and promote the growth of the cancer cells. The role of.5.NET-1 is an effective target for HCC intervention. This study provides a theoretical and experimental basis for further development of NET-1 shRNA for clinical treatment.

【学位授予单位】：南通大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R735.7

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