| Hepatocellular carcinoma(HCC) is one of the most prevalent malignant diseases in China. As has been widely reported, hepatitis B virus(HBV) plays an important and extensive role in the pathogenesis of HCC. HBV-encoded x protein(HBx) is the main factor which involved in the replication of HBV and the progression of HCC induced by HBV. In recent studies, it has been demonstrated that micro RNAs(mi RNAs) played an important role in the pathogenesis of HCC. Studies have reported that HBx can influence some mi RNAs expression in hepatocyte, which resulted in the carcinogenesis of normal liver cells. But, the expression change of global mi RNAs induced by HBx protein is absolutely unknown. Here, in this article, we for the first time comprehensively analysis the global changes of mi RNA expression induced by HBx protein and the potential pathways regulated by these mi RNAs in the pathogenesis of hepatocellular carcinoma.Part I The establishment and identification of HBx-expressed cell linesObjective: To establish HBx-expressed human liver cell lines, transducted HBX gene into L02 and Hep G2 cell lines through lentivirus vector integrated HBX gene.Methods: 1. According to data in Gen Bank, we acquired the full-length sequence of HBX gene and designed the specific primers for cloning it. After the PCR amplification of HBX gene sequence, we cloned it into p ENTR/D-TOPO plasmid through the TOPO cloning reaction and constructed the lentivirus vector p Lenti-HBX through the LR recombination reaction. Then, the p Lenti6.3-HBX vector was co-transfected into 293 T cell lines with the lentivirus packaging system. After the separation and determination of the titer of the lentivirus, subsequent the human liver cell lines L02 and Hep G2 were transfected with this lentivirus. 2. We seed the human normal liver cell line L02 and the human hepatocarcinoma cell line Hep G2 into 24 wells culture plate, until the cell is completely adherence and the attachment rate is about 70%-80% area of the bottom, the cell was transfected by lentivirus. 24 h after transfection, we use the blasticidin to screen out positive-transfected cells and sub-culture it. 3. The expression of HBX in human liver cell lines transfected with lentivirus was identified by two biological methods including relative quantitative RT-PCR and immunohistochemistry(IHC) of cell slides.Results: 1. The lentiviral expression plasmid p Lenti6.3-HBX was successfully constructed, and the HBX gene sequence was confirmed by PCR and sequencing. The p Lenti6.3-HBX plasmid and empty plasmid p Lenti-Control were separately transfected into 293 T cells with Lentiviral Packaging Mix to conduct lentivirus package. The lentivirus Lenti-HBx and Lenti-Control were purified and the titer of lentivirus was detected. 2. We transfected the lentivirus vector Lenti-HBx, and the lentivirus vector Lenti-Control packaged with blank plasmid serve as negative control, into L02 and Hep G2 cell lines, respectively. Screening the positive transfected cells with the Blasticidin in 1ug/ml and sub-culturing them. 3. RT-PCR results shows that, compared with the negative control cells L02-Control and Hep G2-Control, the m RNA levels of HBx significantly increased about 6000 times in the L02-HBx and Hep G2-HBx cells. In addition, the IHC results also showed HBx protein are significantly expressed in L02-HBx and Hep G2-HBx cells, but not in the negative control cells L02-Control and Hep G2-Control.Conclusion: We have constructed the p Lenti6.3-HBX lentiviral expression plasmid and packaged into lentivirus successfully. After transfection of liver cell lines, we constructed L02-HBx and Hep G2-HBx cell lines, and the expression of HBX has been verified in m RNA and protein level.PartⅡ The bioinformatics analysis of the potential biological function of mi RNAs induced by HBx proteinObjective: Screening of aberrant expressed mi RNAs induced by HBx protein, and analyzing of the potential pathways, which resulted in hepatocarcinoma pathogenesis, of these mi RNAs.Methods: 1. Trizol method was used to extract the total RNA from human liver cell lines, L02-HBx, L02-Control, Hep G2-HBx and Hep G2-Control. The mi RNA microarray analysis was carried out by Shanghai Kang Cheng biological company. 2. Three mi RNA target gene prediction programs, Miranda, Microcosom and Target Scan, are used for mi RNA target gene analysis, and we also performed Gene Ontology(GO) analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway analysis for mi RNAs and its target genes. 3. Using relative quantitative RT-PCR for further identification of some mi RNAs and its target genes’ expression changes in cell lines transfected with HBX gene and hepatocellular carcinoma tissues.Results: 1. The total RNA of L02-Control, L02-HBx, Hep G2-Control and Hep G2-HBx were extracted to perform the microarray analysis, and showed that there are 19 mi RNAs expression have more than 2 times of significant changes in L02 and Hep G2 after transfected with Lenti-HBX.2. Mi Randa, Microcosom and Target Scan were used to predict the 19 mi RNAs target genes found in the above, and predicted 38187, 9684, and 5459 target genes respectively, 304 of which were predicted in all three prediction programs. Through GO analysis and KEGG pathway analysis, these mi RNAs and its target genes are proved to be closely related to cellular metabolism, signaling transduction and many human cancers progress. 3. RT-PCR results showed that, in HBx-expressed Hep G2-HBx and hepatocarcinoma samples, the changes of the expression level of mi RNA-663 a, mi RNA-211 and mi RNA-219 is consistent with mi RNA microarray result. In addition, the expression of mi RNA-663 a target gene ACSL3 was down-regulated and the expression of mi RNA-211/mi RNA-219 target gene YY1 was up-regulated.Conclusion: 1. We for the first time analyzed the global changes of mi RNAs expression induced by HBx protein in human hepatocyte, and proved that HBx protein regulate many mi RNAs expression. 2. Through KEGG pathway and GO analysis, we found these aberrant expressed mi RNAs induced by HBx protein is closely related to cell metabolism, cellular signal transduction, hepatitis B process and human tumor pathogenesis. HBx protein maybe promotes HCC development through these mi RNAs co-regulated process. |
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