Carcinogenesis And Mechanism Of Mutant HBx In The Genesis And Development Of HCC

[Background] More than 240 million people worldwide suffer from chronic hepatitis B infection.Each year about 600 thousand people die of liver disease caused by HBV infection.Notably,in Asia and sub Saharan Africa,the prevalence of HCC was significantly higher than that in other regions due to the prevalence of HBV infection.The most dangerous factors of HCC is HBV infection.As the smallest encoding protein of HBV,HBx has trans-activator functions.Through protein-protein interaction process,it can stimulate viral replication or alter host gene expression,thus to promote the occurrence and development of HCC.Because HBV virus has a feature that lack of correction function during replication process,it prefers to have mutations.In fact,HBx gene mutations are often found in HCC patients,which are gradually accumulated during the development of HCC.The common HBx gene mutations associated with HCC include G1613 A,C1653T,T1674C/G,T1753 V,T1768A,and A1762T/G1764 A double mutation.Transfection of HBx with these mutant HBx can promote cell proliferation and migration.However,current studies investigating HBx mutation functions are usually proceed from a single molecule or signaling pathway.It lacks researches illuminating how HBx gene mutations could promote HCC through inflammatory and oncogenic signaling networks via bioinformatics method.[Objective] In this study,we used bioinformatics to study how mutant HBx can develop HCC through altering the inflammatory or oncogenic signaling networks,which helps to find new targets for HBV-HCC diagnosis and therapy.[Method] Firstly,the sequences including wild-type,mutant and C-terminal truncated HBx were amplified from HBV DNA extracted within HCC patients' serum and then constructed into the lentivirus and the Sleeping Beauty transposon plasmids.Reconstructed lentiviral vector and the Sleeping Beauty(SB)transposon system were then used to successfully express HBx protein in hepatocellular carcinoma HepG2 cells and SB mice.By detecting the proliferation,migration and invasion of HepG2 cells,we identified the carcinogenic potency of mutant HBx in hepatocellular carcinoma cells.By detecting the expression of cancer and inflammatory factors in mice,we analyzed the proinflammatory and carcinogenic ability of mutant HBx in vivo.Secondly,signal-to-noise ratio was calculated for gene sets enrichment in HTA2.0 chip analysis.With genes enriched by GSEA,protein-protein interaction was drawing.Within the densest part of the networks,the hub genes were found,which differentially expressed between HBx mutant and wild-type HBx group.In addition,differential expressing genes obtained by comparing mutant HBx and wild-type HBx group both in HTA2.0 and Agilent microarrays were used for GSEA gene sets enrichment.Same GSEA sets in HTA2.0 and Agilent microarray were listed to find inflammatory signaling pathways conducted by mutant HBx gene.In the end,the candidate inflammatory signaling pathways and hub genes were identified in vitro and in vivo in order to find the key genes regulated by mutant HBx.[Results] Mutant,C-terminal truncated and wild-type HBx fragments were amplified from the HBV DNA extracted within HCC patients' serum via PCR method,and then constructed into lentivirus and Sleeping Beauty plasmids.Mutations associated with HCC or cirrhosis are listed below:M1 mutant:A1727G + A1762T/G1764A;M2 mutant:T1753A + A1762T/G1764A;M3 mutant:A1762T / G1764A;M4 mutant:C1653T + T1674 G + A1762T/G1764A C-terminal truncated(Ct-HBx): the G?A mutation was occurred at nt.1733 site,resulting in the HBx truncation since TGG changed into TGA.Firstly,all mutant,truncated and wild-type HBx proteins were successfully expressed in HepG2 cells via lentivirus system.Secondly,the cell cycle,proliferation,migration and invasion experiments showed that compared with wild-type HBx,mutant HBx can accelerate cell cycle progression and promote cell proliferation.The percentage of S pahse HepG2 cells expressing M3,M4 and C-terminal truncated HBx was higher than that of wild-type HBx(p=0.016,0.006 and 0.024 respectively).The proliferation ability of HepG2 cells expressing M4 mutant HBx was significantly higher than that of wild-type group or empty vector group(p values were <0.001 and 0.001,respectively).Tumor formation experiment in nude mice showed that the tumor weight in M4 mutant group was significantly higher than that of wild type mice(p=0.039).2.Utilizing Sleeping Beauty transposon system,mutant,C-terminal truncated and wild-type HBx proteins were successfully expressed in SB mice liver with Fah gene deficiency.The results showed that the tumor growth rate and tumor size in the mutant and truncated groups were higher than that in the wild type group.The cancer occur rate in M4 mutant group and truncated group was significantly higher than that in wild type and empty vector group(M4 vs.Wt,p=0.010;M4 vs.Vector,p<0.001;Ct vs.Vector,p=0.019).The ratio of tumor vs.liver weight in mutant group was higher than that in wild type and empty vector group.The M4 mutant group was significantly higher than that of the wild type group as well as the empty vector group(p values were 0.012 and 0.005,respectively).At the same time,the level of serum inflammatory factors in mice was detected.The expression level of IL-6 in the M4 mutant group was significantly higher than that in empty vector group(p=0.024).The expression level of IL-5 in M4 mutant group was significantly higher than that in wild type and empty vector group(p = 0.010 and 0.043,respectively).The expression level of IFN-? in M4 mutant group was significantly higher than that in wild type and empty vector group(p values were 0.029 and 0.005,respectively.The detection rates of IL-1,IL-12p70,IFN-? and IL-4 were both higher in mutant group than that in wild type group or empty vector group.3.Signal to noise ratio was calculated in HTA2.0 for GSEA enrichment.Gene enriched by GSEA method were then used for protein-protein interaction construction.In the most dense protein-protein interaction module,via comparing between mutant HBx and wide type HBx group,we found the differential hub genes,such as CDC20,PAI-1.Differential genes were used for GSEA enrichment in both HTA2.0 and Agilent microarray.Same GSEA sets in HTA2.0 and Agilent microarray were listed,such as IL-6 inflammatory signaling pathway and partial cancer associated signaling pathways.4.Via RT-qPCR experiment,we found that the CDC20 mRNA level in mutant group was significantly higher than that in wild type group in HepG2 cells(M3 vs.Wt,p=0.026;M4 vs.Wt,p<0.001;Ct vs.Wt,p<0.001).Same results were found in PAI-1 mRNA(M4 vs.Wt,p=0.001;Ct vs.Wt,p<0.001).Detection of the protein level showed that the mutant HBx could increase the expression of CDC20 and PAI-1 when compared with wild type HBx.The result was also confirmed by immunohistochemistry experiment carried out in mouse liver slice.The expression of IL-6 in the M4 mutant group was significantly higher than that in the wide type group or empty vector group(p=0.083 and 0.024,respectively).These results demonstrated that mutant HBx could promote the activation of IL-6 and the expression of CDC20 and PAI-1 compared with wild type HBx.5.Compared with wide type HBx,M4 mutant HBx can promote cell proliferation significantly.When knocked down CDC20 or PAI-1,no difference was found in cell proliferation between HepG2 cells that transiently transfected with plasmids containing M4 mutant HBx or wide type HBx.Thus,we concluded that mutant HBx accelerates cell proliferation via promoting CDC20 and PAI-1 gene expression.[Conclustion] Compared with wild type HBx,mutant HBx has a stronger ability to promote inflammation,cell proliferation and carcinogenesis.In addition,compared with wild type HBx,mutant HBx accelerates cell proliferation as well as carcinogenesis via promoting CDC20 and PAI-1 gene expression.