Study On The Characteristics Of HBV S Gene Mutation In Patients With Occult Hepatitis B Virus (HBV) Infection And The Tumorigenicity Of The Mutant Virus Strain

Objective: To study the mutation characteristics of the major hydrophilic region(MHR)of hepatitis B virus(HBV)S gene in patients with occult HBV infection(OBI)and the tumorigenicity of the mutant virus strain.Methods: A retrospective study was performed on 7,323 patient samples in the serum bank of the Research Center for Clinical Medicine of the Fifth Medical Center of the PLA General Hospital from September 2011 to March 2016.The detection rate of OBI and the characteristics of the HBV S gene MHR mutation were analyzed.And a new mutant virus strain(aa126-127 "RPMNCTI" insertion)from a representative OBI patient was selected.And HepG2 cells were infected by plvx-puro lentivirus expression vector,and the mutant stable cell lines were selected by puromycin,and the wild and empty vector control stable cell lines were screened by the same method.The expression of the protein was analyzed by Western Blot.The clone formation ability and proliferation ability were compared between the mutant group and the wild and blank control group by clonal formation assay and CCK-8 assay.Using the wound healing assay and transwell invasion test to compare the difference in migration ability and invasion ability between the mutant group and the wild and blank control group.Cell cycle and intracellular ROS levels in the mutant group were analyzed by cell cycle assay and ROS assay.Results: Serum HBsAg and HBV DNA were detected in 7,323 patients,among which 14 OBI patients with negative serum HBsAg and positive HBV DNA were screened,and the OBI detection rate was 0.191%(14/7,323).Nine types of MHR mutations were detected in 14 patients,and all the mutations were documented OBI related mutations,except for 126-127 “RPCMNCTI” insertion and F161 S.The results of tumor phenotype experiments showed that,the ability of cell proliferation,clone formation and migration in the mutant group were significantly increased compared with the control group and the wild group.Conclusions: The findings showed that there was a high OBI detection rate in the liver disease population,and literatury-reported-OBI related mutations could be detected in most OBI patients.The new mutation virus strain(aa126-127 "RPCMNCTI" insertion)of HBV S gene has biological characteristics of promoting tumor growth,which might increase the potential risk of progression to liver cancer in patients with chronic HBV infection.