Selection Of HBV CccDNA Mutation In Liver Tissues Of HBV Related HCC And Functional Analysis

Certain HBV viral genome mutations were strongly associated with hepatocellular carcinoma(HCC),and were considered to be the virological factor for HCC,but such hypothesis was mainly based on the observation of higher frequency of certain viral mutations in serum specimens derived from HCC patients,as compared to serum samples at different stages of ASC,CHB,or LC.Because of the proofreading deficiencies of HBV polymerase,the frequency of HBV mutations was increased with the disease progression.So it had been doubted that HBV mutation was the causative factor for HCC,or it only simply reflected the accumulation with prolonged time of disease progression from ASC to HCC.In order to avoid the bias caused by viral evaluation time variant of different stages of HBV infection related disease,in this project,HBV cccDNA in tumor and paired non-tumor tissues of HCC patients were direct sequencing,compared the difference of HBV cccDNA mutations in tumor and paired non-tumor tissues,or higher frequency mutations in tumor tissues,and compared them with viral mutations derived from serum samples of HBV infection-related diseases from ASC to CHB,LC and HCC.Then HBV cccDNA mutations and clinical pathological characteristics of patients(such as HBV DNA quantification,AFP,BCLC,etc.)and survival time after surgery were statistically analyzed to evaluate the clinical relevance of HBV cccDNA mutations,and the possibility of their participation during the course of the development of HCC.Finally,according to the underlying biological characteristics of different mutations,the corresponding function experiments and carcinogenic mechanism tests were conducted.In this study,the levels of HBV cccDNA and total DNA in 29 cases of HCC patients with paired tumor tissues and non-tumor tissues were detected through real-time PCR,via rolling cycle amplification(RCA)and PCR sequencing,we characterized the cccDNA mutation in tumor tissues with paired non-tumor tissues as control.The clinical relevance of HBV cccDNA mutations and clinical pathological characteristics of patients(such as HBV DNA quantification,AFP,BCLC,etc.)and survival time after surgery were statistically analyzed.The results of the study are as following:(1)In this study,HBV cccDNA sequences were successfully collected from 25 cases of tumor tissues and 29 cases of non-tumor tissues.There were 19 cases of paired tumor tissues and non-tumor tissues,except 1 case was infected by HBV genotype B,other 18 cases were infected by HBV genotype C,and 23 cases of HBV cccDNA sequences were HBV C genotype.We compared the sequence variants of HBV ccc DNA among the 23 cases infected with HBV genotype C with wild-type.In hot spots,there were 233 mutation sites which mutation rate were more than 10%.The levels of HBV cccDNA and total DNA in 29 cases of paired tumor tissues and non-tumor tissues were detected.(2)HBV cccDNA mutaions in 23 cases of tumor tissue was statistical analyzed with clinical data of patients(postoperative survival time,BCLC classification,preoperative AFP concentration,etc.)and HBV DNA levels.Kaplan–Meier curves revealed a better overall survival(OS)of patients with T1719 than those with G1719(p=0.032).Concordantly,patients with G1719 preferred to be in high BCLC stage,which implied a worse hepatic function.Additionally,in vitro analysis indicated that G1719 mutation could reduce viral replication efficacy.In line with this,patients with G1719 mutation had relative lower ratio of total DNA/cell and cccDNA/cell than those with T1719.And patients with A1913 had higher relative expression of intrahepatic HBV cccDNA /cell than those with C1913.Of note,the variation of C1329 A and T3098 C implicated better postoperative survival.Finally,statistical significant higher preoperative AFP was found in patients carrying G1078 T,C1653T,G1727 A,C1913A,T1978 C,or C3116 T mutations at the cccDNA level.Cox multi-factor analysis showed that T1719 G mutation was the independent danger prognostic factors,while T3098 C mutation was the independent protection prognostic factors.(3)G588C mutation was only found in 3 cases of tumor tissues.The level of HBsAg in cell lysate in G588 C mutation group was more than that in wild type group(p<0.001),while the level of HBsAg in supernatant in G588 C mutation group was less than that in wild type group(p<0.001),the sum of the level of HBs Ag in supernatant and cell lysates of G588 C mutation group had no difference with that in wild-type group(p>0.05),but the ratio of HBs Ag in supernatant and cell lysates was less than that in wild type group(p<0.001),and the level of HBV DNA in supernatant in G588 C mutation group had no difference with that in wild-type group(p>0.05).The results suggested that G588 C mutation affected the secretion of HBsAg,which might be associated with the development of HCC.(4)In Huh7 and Hep G2 cells,the HBsAg and HBeAg contents in supernatants,the levels of HBV total RNA and 3.5kb RNA in cell lysates,and HBV DNA level in supernatants in HBV 1.2×-T1719 G mutant group were lower than those in HBV 1.2× wild-type group(p<0.001).In Huh7,HepG2,SK-Hep1 and HEK293 T cells,the EnhII activity in HBV 1.2×-T1719 G mutant group were lowed than that in HBV 1.2× wild-type group whether cotransfected with HNF3? plasmid or not(p<0.001),which suggested that T1719 G mutation might reduced the HBV Enh II activity and affected the transcription regulation function of HNF3? on HBV Enh II activity.ChIP-PCR test verified that T1719 G mutation impaired the binding forced between HNF3? and HBV Enh II,the binding forced in G1719 mutant group was lower than that in T1719 wild-type group.And in Huh7 and HepG2 cells,when HBV 1.2× and HBV 1.2×-T1719 G plasmids were co-transfected with HBx plasmid,respectively,the differences of HBsAg,HBeAg,and HBV DNA level in supernatants between the two groups had no statistical significance(p>0.05),whereas,when co-transfected with HBx-mut plasmid,levels of HBsAg,HBe Ag in T1719 G mutant group were still reduced in T1719 G mutant group compared with wild-type(p<0.001),and the levels of HBsAg,HBeAg and HBV DNA in supernatants in HBV 1.2×-T1719 G cotransfected with HBx-mut plasmid group were lowed than that co-transfected with HBx plasmid group(p<0.001),while there were no difference when co-transfected with HBx plasmid.It's worth noting that HBV DNA levels in supernatants were up-regulated whether co-transfected with HBx or HBx-mut plasmid(p<0.001).The results suggested that T1719 G mutation could reduce HBV replication capability,the alteration of HBV Enh II activity and HBx protein both played important role.In conclusion,some cccDNA mutations which had the trend of differences in tumor tissues and non-tumor tissues might be important virological factors for HCC developement.Some cccDNA muations had clinical relevance,G588 C mutation affected the secretion of HBsAg,T1719 G mutation was relevant with BCLC grade and survival time after surgery,and it was an independent danger factor for prognostis prediction of HCC patients after operation.T1719 G mutation could reduce HBV replication capability.