Mechanism Of Hepatitis B Virus Large S Gene And Its Mutations In Promoting The Development And Progression Of HCC

BackgroundAccording to the global tumor registration data,hepatocellular carcinoma(HCC)was the fifth most frequently diagnosed malignancy among males and ninth among females,and the second leading cause of cancer death among males and sixth among females.It is estimated that approximately 50%of HCC new cases and deaths worldwide occured in China.Most HCC cases in China were caused by chronic hepatitis B virus(HBV)infection.As a large population was chronicly infected by HBV,HBV is the main cause of high HCC incidence.The large S region of HBV(preS1/preS2/S),which encodes the surface protein of the virus particles,is a key region for causing viral infection of hepatocytes and inducing HCC occurrence and development.Previous epidemiological data suggest that the variation of the preS region of HBV genome is a risk factor for the development of HCC.At present,it remains to be elucidated which high-risk preS mutation promote carcinogenesis and what the latent mechanism is.PurposeThe aim of this research is to confirm the risk factors of HCC occurrence in chronic hepatitis B(CHB)patients,to clarify what kind of mutations in HBV large S region can promote the occurrence of HCC,to elucidate how these mutations affect the occurrence and development of HCC in virto,to verify the inflammatory and carcinogenic effects of S sequence and mutations,and to provide theoretical bases for further studies on the the prophylaxis of HCC in CHB patients and regarding treatments.MethodsA cohort of 2114 patients with chronic HBV infection was established.Specific primers were designed to amplify the HBV preS region in the peripheral blood of these patients.The amplicons were sequenced by the method of Sanger sequenceing to determine the PreS mutations after alignments.information including sociodemographic data,clinical information and treatments were involved in univariate and multivariate COX regression survival analyses to determine the risk factors of HCC occurrence and and liver disease-related death.Wild-type and three variant HBV large S fragments were used to construct lentiviral vectors,which were transfected into the HepG2 cell line.The effects of related gene sequences on the malignant phenotype of the cells were determined by experiments including cell proliferation,cell migration,cell invasion,and subcutaneous tumor-bearing in nude mice.RNA expression profile microarray was conducted to compare the expression profiles downstream genes regulated by wild type large S gene and mutated ones.Reverse transcription quantitative PCR(RT-qPCR)and western blot were conducted to further study the potential regulatory mechanism of target genes.Sleeping beauty related vectors were constructed with the relevant large S gene sequences and expressed in the mouse liver by tail vein injection,to elucidate the occurrence of hepatic inflammation and hepatocellular carcinoma in mice induced by variant large S gene.The expression profiles of downstream genes of the wild type large S gene and mutated ones were compared by the mouse liver RNA microarray analysis.Immunohistochemistry was conducted to verify the expression level of the target molecules and to further elaborate their mechanism of action.ResultsIn the first part,the cohort study showed that among the 2114 patients with chronic HBV infection,the median follow-up time was 8.92 years.During 18,406 person-years of follow-up,a total of 209 patients developed HCC,with an incidence rate of 9.89%(209/2114).COX regression analysis showed that male gender,old age,cirrhosis,HBV C2sub-genotype,no anti-virus treatment,high direct bilirubin(>7mmol/L),AFP positivity,low albumin(<35g/L),and low platelet count(<100×10~9g/L)were associated with the occurrence of HCC(P<0.05).In addition,C3116T and T31C mutations in the HBV large S region also promote the development of HCC.In CHB patients,sub-genotype C2 was the major gene subtype(73.1%),accounting for 83.3%(125/150)of HCC occurrence.We found HBV C2 subtype related preS mutations,such as G2950A,G2951A,A2962G,C2964A,A3054T,C3063G,T3069G,and A3120T,affected the occurrence of HCC.The combo mutation,such as G2950A/G2951A/A2962G/C2964A(mutation 1),promoted the development of HCC(hazard ratio,[HR]2.51,95%CI[confidence interval],1.10-5.74;P=0.030).Combo mutation C3116T/T31C(mutation 2)also increased the risk of HCC in patients with chronic HBV infection(HR 1.57,95%CI,1.07-2.31;P=0.022).In addition,preS2 deletion(>5bp)promoted HCC in patients who received antiviral treatment(HR2.81,95%CI,1.27-6.22;P=0.011).In the second part of this study,HBV large S fragment with preS2 deletion significantly improved cell proliferation,soft agar cloning,and subcutaneous tumor-bearing ability in nude mice,compared with the wild-type group(P<0.05).However,three variant large S sequences(mutation 1,mutation 2 and deletion)did not increase cell migration or cell migration ability in vitro(P>0.05).After HepG2 cell lines were transfected by target gene,mRNA expression microarray results showed that the wild-type large S fragment mainly affects the biological processes such as intracellular gene transcription,cell cycle,and apoptosis.Compared with the wild type group,mutation 1,mutation 2 and deletion groups all decreased the expression of apoptosis,intracellular immune-related genes,and upregulated the cancer-related signaling pathway.The result of gene quantification also confirmed that the expression of apoptosis promoting genes such as OLR and PMAIP1 in wild type was higher than that in 3 mutation groups,and that expression of apoptosis inhibiting genes,such as BIRC3 and BIRC2 was higher in 3mutation groups than that in wild type group.The expression level of cancer related genes,such as STAT3,was higher in the deletion group than that in the wild type group(P=0.027).The results of western blot showed that the expression level of STAT3 in the three mutation groups were higher than that in wild type group.But there was no significant difference in the expression of phosphorylated STAT3.Under the effect of STAT3 inhibitor,stattic,cell proliferation in each group tended to be consistent.However,under the influence of IL-6,an action of STAT3 signaling pathway,the cell proliferation ability of each group was significantly increased and the difference between groups increased.Correlation analysis showed that the three variant large S genes and inflammatory cytokines promoted the cell proliferation through IL-6/STAT3 signaling pathway.Through the constructed mouse model,three kinds of mutant large S gene were found to reduce the survival of mice.The incidence of tumor for three mutation groups(mutation1,mutation 2 and deletion)were significantly higher than in the wild type,which were36.4%(4/11),57.1%(4/7)and 66.7%(4/6),respectively(P trend=0.023).Liver morphology and H&E staining showed that the expression of HBV large S gene contribute to mice liver inflammation,liver cells showed edema and degeneration,and the characteristic structure of liver was disappear.Immunohistochemical score of HCC related specific molecules CK18,cell proliferation protein Ki-67 and PCNA in mutation 1,mutation 2 and deletion groups were significantly higher than those in wild-type group,respectively(P<0.05).The mRNA expression microarray results of mouse liver showed that the expression of wild-type large S gene regulated apoptosis,immune stress and activation of cancer related signal.The expression of mutation 1,mutation 2 and preS2deletion compared with wild-type large S genes further increased the inflammation and cancer related gene sets,and the activation of cancer related signaling pathways in murine liver cells.The related molecules ELISA results in peripheral blood of mice showed that IL-5,IL-6 and IL-12 were expressed in wild type group were higher than those in vector group,while the three mutation groups were higher than those in wild type group.In addition,the scores of STAT3 and p-STAT3 in three variants of large S gene were higher than those in vector group or wild type group(P<0.05).It shows that three kinds of large S gene mutation,especially preS2 deletion,can activate the IL-6/STAT3 related signal pathway together with the inflammatory state,and then promote the occurrence and development of HCC.ConclusionIn this study,the cohort study confirmed that many clinical indicators and HBV gene preS mutations are risk factor for HCC in CHB patients.It was further found that the combo mutation(G2950A/G2951A/A2962G/C2964A,C3116T/T31C and preS2 deletion)had the effect of promoting HCC in CHB patients.In vitro experiments,it was confirmed that the three variant large S genes,especially the preS2 deletion sequence can upregulate STAT3 signaling pathway,and promote the development of HCC.In mouse model,it was also confirmed that the expression of three mutant large S genes can promote inflammation and cancer in mice liver.This study explains the mechanism of preS-related mutations of HBV large S gene for carcinogenesis,and provides a new idea for preventing and controlling the occurrence of HCC.