| Background At present,there are about 257 million people with chronic hepatitis B virus(HBV)infection in the world,accounting for 3.5% of the world's population.At least 20% of people with chronic HBV infection will develop end-stage liver disease,such as cirrhosis(Liver cirrhosis,LC)or Hepatocellular carcinoma(HCC).Although great achievements have been made in the prevention and treatment of HBV infection in China,the situation is still grim.At present,there are still about 78 million people with chronic HBV infection in China,and about 300,000 people die each year from HBV-related LC or HCC,accounting for 37% to 50% of the global death toll,which has brought about public health in China A great burden.HBV is a hepatotropic virus with a partially relaxed double-stranded circular DNA(rc DNA)genome.The genome is approximately 3.2 kb in length and contains four partially overlapping open reading frames(ORFs),namely the pre-S / S region(nt2848 ~ nt835),the pre-C / C region(nt1814 ~ nt2548),P region(nt2307 ~ nt1623)and X region(nt1374 ~ nt1838),encoding 7 proteins,including 5 structural proteins: core protein(HBc Ag),polymerase protein(Polymerase),hepatitis B surface large protein(LHBs Ag),Hepatitis B protein(MHBs Ag)and hepatitis B surface small protein(SHBs Ag);two non-structural proteins: protein X(HBx)and e antigen(HBe Ag).Due to the lack of a mechanism for correcting mismatched bases by reverse transcriptase during the replication of HBV virus,the genomes of HBV virus strains in the same host are not completely identical,that is,HBV exists as a quasispecies in the host.Under the influence of host immune pressure or drug pressure,HBV quasispecies strains carrying adaptive mutations are selected and continuously replicated to become dominant virus populations,such as in antiviral drugs such as lamivudine(LMV)or adefovir(ADV)Under pressure,the replication of wild-type HBV quasi-population was inhibited,while quasi-populations carrying resistance mutations such as rt M204 V or rt N236 T were selected and continuously expanded into the dominant virus population.HBV mutations have been reported to be related to antiviral resistance,HBV immune escape and disease progression,among which: 1)HBV P region(mainly RT region)mutations are mainly related to nucleoside(acid)analog resistance(NAs);2)Pre-SV mutations in HBV are associated with immune escape of HBV and false negatives in clinical HBs Ag detection;3)Pre-CV mutations in HBV are related with disease progression and pseudo-HBe Ag seroconversion;4)HBV X region mutations are mainly related to the occurrence and development of hepatocellular carcinoma.HBV quasispecies carrying these mutations undergo adaptive expansion in the changing host liver microenvironment,which poses a great threat to the health of infected persons,and also increases the difficulty of antiviral treatment of HBV to a certain extent.With the deepening of people's understanding of HBV gene mutations,related research on HBV mutations has gradually entered the clinic.For example,researchers have developed antibodies against other epitopes to avoid false negative detection of HBs Ag;Region mutation analysis of the causes of NAs resistance;clinicians assess the risk of cirrhosis and hepatocellular carcinoma in CHB patients by detecting pre-C / C region mutations nt G1896 A and HBV core promoter region mutations A1762 T / G1764 A.However,through a review of the previous literature,we found that there are still some problems unresolved: 1)For the HBV mutations in chronically infected people who have not been treated with antiviral therapy and whether there are classic drug-resistant mutations,the results between different studies exist Differences;2)The researchers found that although some RT region mutations are not classical drug resistance mutations,they are closely related to the long-term use of NAs.The above RT region mutations are still unknown due to the unknown drug phenotype;3)At present,the evolutionary characteristics of HBV RT region quasispecies during antiviral treatment and the evolution of pre-C / C quasispecies in disease progression The characteristics have been reported successively,but the characteristics of HBV X region quasispecies during HCC occurrence and development are less reported;4)Although HBV X region and its mutations have been reported to be related to HCC,HBV X region mutations promote the specific mechanism remains unclear.This topic focuses on mutations in the HBV RT region and X region.In order to address the above issues,a series of detailed research work is carried out.Objective 1.By comparing and analyzing the sequence of HBV RT region of untreated chronic HBV infection,it was confirmed whether classical drug resistance was preexisting in chronic HBV infection without NAs,and the distribution characteristics and clinical significance of natural variation of HBV RT region in each structural domain/interdomain of RT region were clarified.2.By comparing the sequence of the HBV RT region in patients with nas-treated CHB with that of untreated chronic HBV infected patients,the characteristics of the nas-treatment-related mutations were clarified,and the in vitro drug-resistant phenotype of the specific mutation rt L229 V found in the study was elucidated.3.By isolating and simulating in vitro the quasispecies of HBV X region in liver cancer tissues and paracancellular tissues,the evolution characteristics of quasispecies of HBV X region in liver cancer tissues and paracancellular tissues were analyzed and compared,and the mutation differences of HBV X region clones in the two types of tissues were compared,in order to clarify the evolution rules of quasispecies of HBV X region in the process of HCC occurrence.4.New HBx triple mutation T81P/S101P/L123 was found in liver cancer tissue in HCC patients prior to this study.In order to clarify its carcinogenic effects,we used to carry HBx-T81P/S101P/L123 S stable-transfection liver cancer cell line,through cell cycle detection,EDU intake,cell concentration,protein immunoblotting and other experiments to clarify HBx-T81P/S101P/L123 S in HCC development of the molecular mechanism.Materials and methods 1.Sanger sequencing was used to detect the HBV RT region sequences in 493 chronically infected HBV patients without antiviral treatment,and 63 secondaries were sequenced at the same time;another 3 were without antiviral treatment HBV chronically infected individuals were only sequenced for the second generation.2.Sanger sequencing was used to detect the HBV RT region sequences in 285 non-antiviral infections and 214 HBV chronic infections who had been treated with nucleoside(acid)analogues for at least 3 months.3.T-A clones were used to simulate the HBV X-domain quasispecies in the liver tissue of patients with HBV-related hepatocellular carcinoma in vitro,and Sanger sequencing was used to detect the DNA sequence cloned for each HBV X-domain.4.Use the Shannon entropy formula Sn =-?i(pi × lnpi)/ ln N(i refers to the type of quasispecies,pi refers to the proportion of the number of quasispecies in the entire quasipopulation,and N refers to the number of all virus strains)Calculate the quasispecies complexity of the HBV X region in the liver tissue;use the biosense software MEGA 7 to calculate the quasispecies diversity of the HBV X region in the liver tissue,including genetic distance(d),number of synonymous mutation Substitutions Per Synonymous Site(d S)and Number of Non-synonymous Substitutions Per Non-synonymous Site(d N).5.The phylogenetic tree of HBV X-domain quasispecies in liver tissue was constructed using MEGA 7 of Shengxin software,and the homology and genotype of HBV X-domain quasispecies were analyzed.6.Using the online tool STRUM,the computer simulates the three-dimensional spatial conformation of rt WT,rt L229 V,rt M204 V,rt M204 V / L229 V,x WT,x L123 S,x S101 P / L123 S,and x T81 P / S101 P / L123 S.7.Use the online tool ENTROPY to analyze the Shannon entropy difference of HBV X region mutations in liver cancer tissues and adjacent tissues.8.Construction of Huh 7 cell lines stably overexpressing HBx-WT,HBx-L123 S,HBx-L123 S / S101 P and HBx-T81 P / S101 P / L123 S using lentiviral vectors.9.Evaluate the effects of HBx-T81 P / S101 P / L123 S on the proliferation of Huh 7 cells using CCK-8 experiments,clone formation experiments,cell cycle detection,EDU incorporation experiments,and Western blotting.10.Evaluate the effect of HBx-T81 P / S101 P / L123 S on the genome stability of Huh 7 cells by direct observation under phase contrast microscope,EDU-APC / PI double staining,apoptosis detection,karyotype detection,and Western blotting.11.Use small interfering RNA to knock down the expression of MYC and evaluate the effect of MYC on the effect of HBx-T81 P / S101 P / L123 S.Results 1.The characteristics of HBV RT region mutations in patients with chronic HBV infection 1.1.Based on the identification of genotype-specific amino acid polymorphisms,we compared the DNA sequences of the HBV RT region of 427 chronically infected HBV patients and found that 156 chronically infected patients carry RT region mutations.These mutations are mainly distributed at 56 amino acid sites.Among them,mutations at the 36 amino acid residue sites caused changes in the epitope of the RT protein or S protein.1.2.After analyzing the mutation frequencies of different domains in the RT region,it was found that the mutations were mainly concentrated in the C domain,E domain,and C-D interdomain.Among them,the frequency of mutations in the RT region of chronic carriers is highest in the C domain;in patients with chronic hepatitis B, mutations are mainly distributed in the E domain and the CD domain;in patients with advanced liver disease,despite the frequency of mutations between different domains There is no significant difference,but the C domain and the CD domain have relatively high mutation frequencies.At the same time,we also found that the frequency of mutations in the C domain of chronic carriers is significantly higher than the frequency of mutations in the C domain of chronic hepatitis B patients.1.3.After comparing the clinical indicators of 271 chronically infected patients with RT mutations and 156 chronically infected patients without RT mutations,it was found that they carry mutations in the RT region,especially those without-ple resistances.People with chronic HBV infection treated with the virus have significantly lower HBV DNA loads,HBs Ag levels,and HBe Ag positive rates.At the same time,their mean age is significantly higher and their risk of liver fibrosis is higher.1.4.We used next-generation sequencing and Sanger sequencing to analyze the DNA sequences in the range of rt201 to rt335 of 66 chronically infected HBV patients without antiviral treatment.We found that within the detection range of Sanger sequencing,the two sequencing methods The results were basically consistent;the second-generation sequencing detected a lower proportion of classic primary drug-resistant mutations in some patients with chronic HBV infection;the second-generation sequencing also found rt251,rt266,rt274,rt280,rt283,rt284,and rt286.The frequency of mutations at points gradually increases as the disease progresses.2.The characteristics and role of HBV RT region mutations in patients with chronic HBV infection treated with nucleoside(acid)analogs 2.1.Among non-NAs-infected patients,the only mutations that differ in frequency between genotypes are rt L199 V and rt L220 I / V;in CHB patients treated with NAs,the mutations that differ in frequency between genotypes are rt A181 T / V and rt L220 I / V,suggesting that genotypic differences have limited effect on RT mutations.2.2.Classic resistance mutations rt L180 M,rt A181 T / V / I,rt M204 I / V / L,rt N236 T / I,and mutations with unclear resistance phenotype rt L229 V / M / F have significantly higher mutation frequency in CHB patients receiving NAs in non-NAs-infected patients,rt L229 V / M / F may affect the antiviral efficacy of NAs.2.3.After reviewing the medication of CHB patients with rt229 mutation,it was found that the rt229 mutation was related to the use of LMV,Ld T and ETV,and many patients had rt229 mutation and rt204 and rt180 mutations at the same time;computer simulation of protein three-dimensional Structural results showed that rt M204 V and rt L229 V significantly changed the spatial conformation of RT protein.2.4.The results of in vitro replication phenotyping experiments showed that rt L229 V significantly reduced the replication capacity of HBV,and the effect of rt L229 V on HBV replication was greater than that of rt M204 V.The results of in vitro drug resistance phenotyping experiments showed that rt L229 V mutations conferred resistance to LMV in HBV.And the simultaneous occurrence of rt M204 V and rt L229 V mutations will greatly reduce the sensitivity of HBV to ETV.3.Characteristics and role of HBV X-region quasispecies in HBV-related hepatocellular carcinoma 3.1.We evaluated the HBV X-region quasispecies heterogeneity from two aspects of complexity and diversity,and found no significant difference in the HBV X-region quasispecies diversity in liver cancer tissues and adjacent tissues;however,within liver cancer tissues,The quasispecies complexity of the HBV X region is significantly lower than that of the adjacent tissues,suggesting that there are differences in the HBV X region quasispecies heterogeneity between liver cancer tissues and adjacent tissues.3.2.By constructing a phylogenetic tree of HBV X region quasispecies in liver tissues,we found that the evolution of HBV X region quasispecies in liver cancer tissues and the evolution of HBV X region quasispecies in adjacent tissues have a significant difference,suggesting that liver cancer cells and the The microenvironment of adjacent liver cells is different.3.3.Through cloning and sequencing of patients with hepatocellular carcinoma,we found a mixed infection of B and C genotypes;in patients with mixed infection,the C genotype HBV strain tends to appear in liver cancer tissues,while the B genotype HBV virus Tendency to appear in adjacent tissues.3.4.Second-generation sequencing results show that mixed genotype infections are common in the population.Among them,the proportion of HBV strains with genotype B in chronic carriers is slightly larger than that with genotype C.The proportion of HBV strains with B genotype in chronic hepatitis B population is significantly larger than that with C genotype.Genotype,suggesting that the mixing ratio of different genotypes is related to disease progression.3.5 The load of HBV DNA of C genotype is significantly higher than the load of B genotype in the immune tolerance stage.The HBV DNA load of patients with B genotype in the immune clearance stage is slightly higher than that of C gene patients.The HBV DNA load is slightly higher than that of patients with genotype B genotypes,which indicates that the change trend of HBV DNA load of different genotypes is more consistent with the change trend of its mixing ratio in the population.3.6.By comparing the frequency of mutations at amino acid sites(Figure 4A)with Shannon entropy(Figure 4B),we found that mutations at 18 amino acid sites were significantly different between liver cancer tissues and adjacent tissues;of these,12 There were more mutations in the liver cancer tissues,and there were 6 more mutations in the adjacent tissues.The results of three-dimensional structure computer simulation showed that the spatial conformation of HBx-T81 P / S101 P / L123 S was higher than that of wild type HBx Significant changes have taken place.4.The mechanism of HBx-T81 P / S101 P / L123 S in the occurrence and development of HCC 4.1.Cell colony formation experiments showed that Huh 7-Lv TM formed more cell colonies in vitro;CCK8 experiment showed that Huh 7-Lv TM increased the number of cells significantly more;EDU incorporation test showed that more Huh 7-Lv TM cells are in the DNA replication phase;cell cycle tests show that more Huh 7-Lv TM is in the S phase;Western-Blot experiments show that protein levels of E2F1 and p-Rb are significantly increased in Huh 7-Lv TM.The above results suggest that HBx-T81 P / S101 P / L123 S can promote the proliferation of Huh 7 cells.4.2.Through phase-contrast microscopy,we found that multinucleated giant cells were formed in Huh-Lv TM;using EDU-APC / PI double staining,we found that significantly more Huh 7-Lv TM formed more than tetraploid genomes in G2;Under the action of genotoxic drugs,more Huh 7-Lv TM cells undergo apoptosis;Western-Blot experiments showed that the protein levels of CDT1 and ATM in Huh 7-Lv TM cells increased significantly;karyotype analysis results showed that Huh 7-The karyotype of Lv TM has a phenomenon of chromosome breakage.The above results suggest that HBx-T81 P / S101 P / L123 S can cause genomic instability in Huh 7 cells.4.3.Western-Blot experiments showed that Huh 7-Lv TM cells expressed more MYC protein.Under the condition that MYC was knocked down,the results of Western-Blot experiments,EDU incorporation tests,and cell cycle tests showed that changes in MYC levels had a significant effect on Huh 7-LvTM.Conclusions 1.The characteristics of HBV RT region mutations in patients with chronic HBV infection In this study,Sanger sequencing and second-generation sequencing were used to confirm that a low proportion of primary drug-resistant mutations existed in untreated HBV chronically infected people;by analyzing the effects of RT mutations on antigenic epitopes and RT mutations and infected persons It is found that immune pressure may be one of the driving forces for the natural variation of the RT region.At the same time,we analyzed the natural variation of the RT region at different stages of liver disease and found that RT mutations may be involved in the progression of liver disease.2.The characteristics and role of HBV RT region mutations in patients with chronic HBV infection treated with nucleoside(acid)analogs In this study,the replication ability of HBV with rt L229 V mutation in vitro was significantly reduced and resistance to LMV was demonstrated for the first time by analyzing RT mutations in NAs treated and untreated populations.When HBV has rt M204 V and rt L229 V divalent mutation,its sensitivity to ETV significantly decreases.More importantly,the above results provide experimental basis for the selection of clinical medication and the evaluation of efficacy.3.Characteristics and role of HBV X-region quasispecies in HBV-related hepatocellular carcinoma In this study,clonal sequencing was used to find that the heterogeneity,evolution pattern,and mutation characteristics of HBV X-domain quasispecies in liver cancer tissues were significantly different from those of HBV X-domain quasispecies in adjacent tissues.The discovery for the first time revealed the characteristics of HBV X-zone quasispecies in the liver tissue of HCC patients,and provided a new idea for understanding the potential carcinogenic mechanism of HBV.4.The mechanism of HBx-T81 P / S101 P / L123 S in the occurrence and development of HCC In this study,through functional verification of HBx-T81 P / S101 P / L123 S stably transformed cell lines,we found that HBx-T81 P / S101 P / L123 S can promote the proliferation of liver cancer cell lines and genomic instability.By detecting the expression levels of related molecules and verifying cell function after MYC knockdown,it was found that changes in MYC levels have a significant effect on Huh 7-Lv TM.Therefore,we believe that HBx-T81 P / S101 P / L123 S regulates the host cell's malignant proliferation and genomic instability mainly through MYC. |
PDF Full Text Request