| Part ?: Expression analysis of ADH1 B in liver cancer and its adjacent tissuesObjective: The purpose of this study was to explore the expression differences of ADH1 B gene in hepatocellular carcinoma(HCC)and adjacent tissues,so as to lay a solid foundation for further understanding of the function and mechanism of ADH1 B gene,and to provide a theoretical basis for further understanding of the pathogenesis and gene therapy of ADH1 B gene in hepatocellular carcinoma(HCC).Methods: RT-qPCR and Western Blot were performed on 10 HBV-related HCC samples collected from clinical operations,and 5 normal liver tissues and adjacent tissues,to analyze the expression differences of ADH1 B gene in liver cancer and the corresponding adjacent clinical samples(P<0.01).Results: The expression of ADH1 B gene in liver cancer tissues was significantly lower than that of adjacent tissues from protein and gene m RNA levels,and the difference was statistically notable.Conclusion: it was preliminarily verified that the expression of ADH1 B gene in HBV-associated HCC liver cancer and adjacent tissues was different.This will lay a solid foundation for the further influence of this gene on the biological behavior of liver cancer cell lines.Part?: Construction of recombinant lentivirus vector overexpressing ADH1 B geneObjective: Recombinant lentiviral vector(LVs)has been used as a gene therapy vector for acquired and inherited diseases,and has become an effective and universalvector for gene transfer to divided and non-divided cells in vivo or in vitro.The purpose of this study was to construct lentivirus expression vectors that overexpressed ADH1 B gene and provide packaging tools for stable cell lines that overexpressed ADH1 B gene.Methods: Primers were designed based on the ADH1 B gene sequence found by Genbank,the ADH1 B gene fragment was amplified by PCR,and the GV-492 vector was digested by Bam HI/Age I at the same time.The GV492-ADH1 B vector plasmid containing puromycin resistance was transfected with p Helper1.0 and p Helper2.0 lentivirus helper plasmid to collect the lentivirus supernatant and determine the lentivirus titer.The overexpressed recombinant lentivirus carrying GFP and ADH1 B genes was obtained.Results: The sequencing results showed that the ADH1 B overexpressed lentivirus vector was successfully constructed by ordinary PCR amplification,enzyme digestion identification,lentivirus quality monitoring and gene sequencing,and the detected virus titer was 2E+9TU/m L.Conclusion: The recombinant lentivirus expression vector overexpressing LV-ADH1 B gene has been successfully constructed,which provides a good tool for the further establishment and stabilization of HCC cell lines and the discussion of the biological function and molecular mechanism of this gene in HCC.Part ?: Effect of ADH1 B gene on the biological function of human hepatocellular carcinoma cell linesObjective: This part mainly explore built and packaged ADH1 B gene expression of recombinant lentivirus stable liver cancer cell lines,using the stable cell lines to observe its biological functions of hepatocellular carcinoma(HCC),and search for potential meaningful HCC diagnosis biomarkers and drug therapeutic targets,in order to further clear clues ADH1 B gene related signaling pathway and direction.Methods: 1.RT-q PCR and Western Blot were performed on normal hepatocytes LO2,HepG2 and Hep G2.2.15 to analyze the expression differences of ADH1 B gene in normal hepatocytes and hepatocellular carcinoma cells.2.A stable liver cancer cell line with overexpression of ADH1 B gene was constructed and identified,and liver cancer cells were used as blank control(BC),transfected with GV-492 empty vector chronic disease virus as negative control(NC),and LV-ADH1 B transfected with liver cancer cells as overexpression group(OE).RT-q PCR and Western Blot were used again to verify the difference in the expression of ADH1 B m RNA and protein in liver cancer cell lines to verify the effect of ADH1 B overexpression in liver cancer cell lines.3.CCK-8,Cell scratch test,Plate cloning formation test,Transwell cell migration and invasion test were used to detect the changes in proliferation,migration and invasion ability of Hep G2 cells after overexpression of ADH1 B gene.4.Regular trains ADH1 B gene expression of stable cell lines(BC)blank group,negative control(NC),express group(OE)Hep G2 and Hep G2.2.15 cells,24 h after collection of groups of cells and add Trizol cracking fluid cracking each cell,inserted in 1.5 ml EP preservation,placed in a bubble with dry ice box sent to Shanghai ou yi company in gene chip detection and analysis.Results: 1.The expression levels of ADH 1B protein and m RNA in Hep G2 and Hep G2.2.15 in liver cancer cells were significantly lower than that in normal liver cells(P<0.01).2.Western Blot and rt-qpcr were used again to compare the ADH1 B protein and m RNA extracted from Hep G2 and Hep G2.2.15 cell lines transfected with recombinant lentivirus lv-adh1 b,and the results showed that:The expressions of ADH1 B protein and m RNA in Hep G2 and Hep G2.2.15 cell lines with overexpression of ADH1 B gene were significantly increased,and the difference was statistically significant(P<0.05),indicating the successful construction of Hep G2 and Hep G2.2.15 cell lines transfected with lv-adh1 b and gv-492 null vector lentivirus.3.Cell function test: CCK-8 assay showed that,compared with the HepG2 blank group(BC)and the Hep G2 negative control group(NC),the Hep G2 group(OE)with overexpression of ADH1 B gene showed lower cell activity,and the difference was statistically significant(P<0.05);Scratch test results showed that after 24 h and 48 h of cell culture,the scratch area of Hep G2(OE)cell line overexpressing ADH1 B gene was significantly higher than that of blank group(BC)and negative control group(NC),and the difference was statistically significant(P<0.01);The results of plate cloning formation experiment showed that compared with blank group(BC)and negative control group(NC),the number of overexpressed ADH1 B genome(OE)Hep G2 cell clonal group was significantly reduced,and the difference was statistically significant(P<0.01);The results of Transwell cell migration and invasion experiment showed that,compared with the blank group(BC)and the negative control group(NC),the number of translocular overexpression of ADH1 B genome(OE)Hep G2 cells was significantly reduced,and the difference was statistically significant(P<0.05).4.The results of chip sequencing on Hep G2 and Hep G2.2.15 cells by overexpression of ADH1 B gene showed that: compared with the control group,Hep G2 with overexpression of ADH1 B gene showed differences in the Pathway of KEGG enrichment in NF-?B ? Alcoholism ? MAPK ? TNF?,etc.Hep G2.2.15 overexpressed ADH1 B gene showed KEGG enrichment Pathway differences in ECM receptor,Ca2+,and Jak-STAT etc.compared with the control group.Conclusion: 1.The difference of ADH1 B gene in normal hepatocytes and hepatocellular carcinoma cell lines Hep G2 and Hep G2.2.15 was preliminarily verified,which was consistent with the experimental results of part I hepatocellular carcinoma.2.Hep G2 and Hep G2.2.15 cell lines expressing ADH1 B gene were successfully constructed.3.It was predicted that ADH1 B gene might inhibit HCC metastasis by inhibiting the proliferation,migration and invasion ability of HCC cells.4.Liver cancer cell lines Hep G2 and Hep G2.2.15 transfected with LV-ADH1 B,showed changes in gene expression in epithelial mesenchymal transformation,alcohol metabolism,cell apoptosis on the signaling pathways. |
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