Analysis Of HBX Mutations In Tissues Of Hepatocellular Carcinoma And Biological Impact Of Different HBX Mutations As Well As Their Mechanism On HepG2 Cell Line

Background and aims Hepatocellular carcinoma（HCC）is one of the most prevalent and lethal cancers worldwide.Chronic hepatitis B virus（HBV）infection has been demonstrated to be a major risk factor.Further research indicates that HBV X（HBX）gene plays a crucial role in hepatocarcinogenesis.Recently,emerging evidence has reported that mutations including point mutations and deletion mutations are frequently detected in HBX gene region,and are closely related to liver cancer.However,the function and mechanism of HBX mutations in the development of HCC have not yet been determined.Endoplasmic reticulum stress（ERS）and autophagy were recently reported to be involved in the progress of HCC.Furthermore,recent studies indicates that ERS and autophagy maybe under the regulation of HBX.However,it is still unknown whether HBX mutations involved in hepatocarcinogenesis through the regulation of ERS and autophagy.Therefore,the present study was conducted to analyze the characteristic of HBX mutations in tissues of HBV-associated HCC,and based on this to investigate the influences of HBX mutations on biological function of Hep G2 cells,as well as further explore the possible mechanism mediated by ERS/autophagy in HBXassociated hepatocarcinogenesis.Methods 1.61 pairs of carcinoma and adjacent pericarcinoma tissues of HBV-related HCC patients receiving surgery between January 2015 and December 2016 were collected from Affiliated Tumor Hospital of Guangxi Medical University.HBX gene was amplified by nested polymerase chain reaction（PCR）from extracted HBV DNA,and then was subjected to Sanger sequencing.Finally,the sequencing data were analyzed by Mutation Surveyor（V4.0.8）software.The sequences acquired in the present study were compared with the reference sequence in NCBI database（Gen Bank accession no.NC₀03977.1）.2.HBX mutations were selected based on the result of part one study,and then they were artificially synthesized followed by clone into the eukaryotic expression vector GV362.Then the HBX-GV362 recombinant vectors were transfected into Hep G2 cells by Lipofectamine 3000,and the GV362 blank vector was used as a control.Finally,Hep G2 cells stably expressing HBX protein were screened by G418 and verified by Western blot.3.The influences of HBX mutations on biological function of Hep G2 cells were studied as follows: cell counting kit-8（CCK-8）assay was exploited to evaluate cell proliferation;colony formation assay was performed to test tumorforming ability;flow cytometry（FCM）was utilize to detect cell cycle and apoptosis;wound healing assay was carried out to assess cell migration.4.Expressions of PDI,Grp78 and CHOP proteins were detected by Western blot for preliminary evaluation of the relationship between HBX and ERS.Expressions of P62,LC3 and Beclin 1 proteins were detected by Western blot for preliminary evaluation of the relationship between HBX and autophagy.Then the present study further verified whether autophagy and ERS were regulated by HBX mutations,as well as whether autophagy interacted with ERS in hepatocarcinogenesis as follows:（1）Stable Hep G2 cells transfected with HBXGV362 recombinant vectors were treated with ERS inducer tunicamycin（TM）and ERS inhibitor 4-Phenylbutyric acid（4-PBA）respectively,then Western blot was performed to detect P62,LC3 as well as Beclin 1 protein expressions,followed by FCM to evaluate cell cycle and apoptosis.（2）Stable Hep G2 cells transfected with HBX-GV362 recombinant vectors were treated with autophagy inducer rapamycin（RAPA）and autophagy inhibitor chloroquine（CQ）respectively,then Western blot was performed to detect PDI,Grp78 as well as CHOP protein expressions,followed by FCM to evaluate cell cycle and apoptosis.5.Statistical analysis.SPSS 16.0 software was used for statistical analysis of the acquired data.Quantitative variables were expressed as mean ± standard deviation（SD）,and the comparison between two groups was performed by t test,while the comparison among multiple groups was performed by one-way analysis of variance（One-Way ANOVA）.Qualitative variables were expressed as rate or composition ratio（%）,and the comparison between groups was performed by using the chi-square test.Statistical significance was considered to be existed when P value <0.05.Results 1.A total of 122 HBX gene sequences were acquired in the present study,and both deletion mutations and point mutations were found.The detailed results were as follows:（1）The distribution of HBX deletion mutations between carcinoma and pericarcinoma tissues showed no statistical difference（P>0.05）, and the distribution of HBX carboxy-terminal deletion mutations between carcinoma and pericarcinoma tissues also showed no statistical difference（P>0.05）.However,the results showed that the deleted length of carboxylterminal was among 12-270 bp and the deleted length in carcinoma tissues was significantly longer than that in adjacent pericarcinoma tissues（33.10±71.45 vs.12.64±23.82,P<0.05）.（2）The number of point mutations in carcinoma and adjacent pericarcinoma tissues were 260 and 190,respectively.A total of 62 hot spot mutations were identified,whereas only 10 indicated statistical difference between carcinoma and adjacent pericarcinoma tissues（P<0.05）.The 10 hot spot mutations were C1653 T,C1655T,C1673 G,T1719G/G1721 A,T1753V（C/A）,A1762 T,G1764A,A1762T/G1764 A,A1775G and T1800 C.Except for A1775 G mutation,the detection rate of other point mutations in carcinoma tissues was statistically higher than that in adjacent pericarcinoma tissues（P<0.05）.2.Based on the findings of the first part study,we selected a total of eight HBX mutation sequences and named them as: HBX-C1479A/A1499 G,HBXT1719G/G1721 A,HBX-A1762T/G1764 A,HBXT1753A/A1762T/G1764A/T1768 A,HBX-del 54,HBX-del 54/mt,HBX-del 105,HBX-del 105/mt.Meanwhile,we also included the wild-type HBX gene sequence（named HBX-wt）.The Hep G2 cells stably expressing HBX protein were successfully established through molecular cloning technique and lipofectin transfection method.The expression of HBX protein was detected in HBX stably transfected Hep G2 cells but not detected in untransfected and empty vector transfected Hep G2 cells.3.According to the result of CCK-8 assay,the proliferation ability of Hep G2 cells stably transfected with HBX-wt or HBX mutations was significantly enhanced compared to untransfected or empty vector transfected cells（P<0.05）.Except for HBX-C1479A/A1499 G and HBX-T1719G/G1721 A,the proliferation ability of Hep G2 cells stably transfected with HBX mutations,especially mutations of HBX-T1753A/A1762T/G1764A/T1768 A and HBX-del 105/mt,were greater than that of cells transfected with HBX-wt（P<0.05）.The colony formation assay displayed the similar results.According to FCM results,Hep G2 cells transfected with HBX-wt or HBX mutations significantly increased the proportion of cells in（G2+S）phase（P<0.05）while significantly decreased the proportion of cells in G1 phase（P<0.05）in comparison with untransfected or empty vector transfected cells.Except for HBX-C1479A/A1499 G and HBXT1719G/G1721 A,the proportion of（G2+S）phase significantly increased（P<0.05）while the proportion of G1 phase significantly decreased（P<0.05）in Hep G2 cells transfected with HBX mutations than that with HBX-wt.Among HBX mutations,the influence of HBX-T1753A/A1762T/G1764A/T1768 A and HBX-del 105/mt was more prominent（P<0.05）.Compared with untransfected or empty vector transfected cells,the apoptotic rate of HBX mutation transfected cells significantly decreased（P<0.05）,whereas that of HBX-wt transfected cells had no significant changes（P<0.05）.Similarly,the impact of HBXT1753A/A1762T/G1764A/T1768 A and HBX-del 105/mt was more prominent（P<0.05）among HBX mutations.According to wound healing assay,only cells transfected with HBX-A1762T/G1764 A,HBXT1753A/A1762T/G1764A/T1768 A,HBX-del 105,HBX-del 105/mt,HBX-del 54 and HBX-del 54/mt significantly raised the scratch healing index（P<0.05）,whereas HBX-wt and HBX-C1479A/A1499 G as well as HBX-T1719G/G1721 A had no significant changes（P<0.05）in comparison with untransfected or empty vector transfected cells.4.The protein expressions of PDI,Grp78 and CHOP were enhanced in HBX mutation transfected Hep G2 cells while that in HBX-wt transfected cells showed no changes compared to empty vector transfected cells.The protein expressions of Beclin 1 and LC3 as well as the expression ratio of LC3-II to LC3-I were elevated in HBX mutation transfected Hep G2 cells while that in HBX-wt transfected cells showed no changes in comparison with empty vector transfected cells.Meanwhile,P62 expression was reduced in HBX mutation transfected Hep G2 cells while that in HBX-wt transfected cells exhibited no changes in comparison with empty vector transfected cells.Based on the above results,Hep G2 cells transfected with HBX-T1753A/A1762T/G1764A/T1768 A and HBX-del 54/mt were selected for further study.The results were as follows:（1）When ERS of the transfected cells was inhibited by 4-PBA,the protein expressions of Beclin 1 and LC3 as well as the expression ratio of LC3-II to LC3-I were down-regulated,while the expression of P62 was up-regulated.Meanwhile,4-PBA treatment resulted in increased proportion of cells in G1 phase（P<0.05）,decreased proportion of cells in（G2+S）phase（P<0.05）and increased apoptotic rate（P<0.05）.When ERS of the transfected cells was induced by TM,autophagy associated proteins as well as cell cycle and apoptosis showed opposite changes.（2）Whether autophagy of the transfected cells were inhibited by CQ or induced by RAPA,ERS associated proteins exhibited no significant changes.Nevertheless,CQ treatment led to increased proportion of cells in G1 phase（P<0.05）,decreased proportion of cells in（G2+S）phase（P<0.05）and increased apoptotic rate（P<0.05）,while RAPA treatment led to opposite changes.Conclusions 1.Although abundant mutations including point mutations and deleted mutations existed in HBX gene sequences,just a few mutations were identified to be associated with HCC.The present study indicated that C1653 T,C1655T,C1673 G,T1719G/G1721 A,T1753V（C/A）,A1762 T,G1764A,A1762T/G1764 A,A1775G,and T1800 C mutations may be associated with HCC.2.HBX gene mutations can promote Hep G2 cell proliferation,inhibit its apoptosis,and promote its migration.In addition,with the exception of the HBXC1479A/A1499 G and HBX-T1719G/G1721 A mutations,the remaining HBX mutations we studied had a stronger effect on the biological function of hepatocellular carcinoma Hep G2 cells than the HBX wild gene.Furthermore,HBX-T1753A/A1762T/G1764A/T1768 A and HBX-del 105/mt mutations had the most significant effect on the biological function of Hep G2 cells.3.HBX mutations could induce ERS and autophagy.Furthermore,HBXT1753A/A1762T/G1764A/T1768 A and HBX-del 105/mt mutations could induce autophagy through ERS,and then resulted in the promotion of cell cycle and inhibition of apoptosis.This may be one of the mechanisms underlying the occurrence and development of HBV-associated HCC.