Hepatitis B Virus Regulates HTERT And ZHX2 Involving Hepatocellular Carcinoma Development

OBJECTIVESHepatocellular carcinoma(HCC) is a kind of malignant tumor that affects human health severely.China is HCC high-incidence district and takes up 55%of HCC cases in the world.HCC incidence in developed countries is increasing in recent years. Chronic hepatitis B virus(HBV) infection has been identified as a leading risk factor for HCC in China.So it is urgent to reveal the mechanisms of HBV related HCC.The mechanism of HBV related HCC is complicated.The integration of HBV-DNA into the host genome can trigger cis-activation directly;Also regulatory proteins HBX and preS2 activators encoded by integrated subviral HBV genomes can exert a tumor promoter-like function,resulting in positive selection of cells producing a functional regulatory protein.To search the key genes regulated by HBV,not only provides newsights into mechanisms of HBV related HCC development,but also gives new potential targets for drug design.Here,two important HCC related genes,human telomerase reverse transcriptase (hTERT) and zinc-finger and homeoboxe 2(ZHX2) were selected to be further studied.It has been proved that telomerase activation is an important event during the process of immortalization and upregulation of hTERT might be a critical event in the development of human cancers,including HCC.Earlier studies from our laboratory have shown that preS2 overexpression can upregulate both hTERT expression and telomerase activity in HepG2 cells,resulting in enhanced cell growth which could be abolished by knocking down of hTERT in the cell,indicating that preS2 involves in HCC via hTERT upregulation.However,what's the mechanism remains unknown.In the first part of the study,cotransfection and report assay were involved to explore the molecular mechanism of preS2-mediated hTERT upregulation.Zinc-finger and homeoboxes 2(ZHX2) gene belongs to a small family of transcription factors that also includes ZHX1 and ZHX3.All ZHX proteins contain two C2-H2 zinc-finger motifs and four homeodomains,are ubiquitously expressed and localized to the nuclei of cells,and appear to function as transcriptional repressors. Insight into the biological function of ZHX2 has come from the analysis of alpha-fetoprotein(AFP) expression in BALB/cJ mice.ZHX2 is reported to be responsible for adult AFP repression in BALB/cJ mice.H19 and Glypican 3 are two additional targets of ZHX2 in the mice liver and have the same expression law as AFP. Our previous studies show that ZHX2 is a repressor of AFP in HCC cell lines,which indicates the potential role of ZHX2 in HCC development.In the second part of this study,we aim to explore if HBV regulate ZHX2 and if ZHX2 is responsible for GPC3 regulation in HCC.In order to investigate the mechanisms of HBV related HCC and gene regulation during HCC development,we focus our studies on the following aspects:1.The precise mechanism for preS2 mediated transcriptional activation of the hTERT gene in HCC development.2.The role of ZHX2 in HBV related HCC:(1) The regulation of Hepatitis B Virus and its viral proteins on ZHX2 expression in HCC.(2) The regulation of ZHX2 on GPC3 expression in HCC and the mechanism. METHODS1.The molecular mechanism for preS2 mediated transcriptional activation of the hTERT in HCC development(1) HepG2.2.15 cells were pre-treated with preS2 antisense RNA to block the expression of preS2,RT-PCR and Western blot were applied to detect the expression change of hTERT.(2) 16 HCC samples were collected,RT-PCR and Western blot were applied to detect the expression of preS2 and hTERT respectively.(3) preS2-EGFP fusion protein eukaryotic expression vector was constructed and named pEGFP-preS2.48h After transfected into HepG2 cells,the cells were stained with 4',6-diamidino-2-phenylindole(DAPI).Digital images were taken from a OlympusⅨ81 fluorescence microscope with 400×magnification.(4) 0.5μg hTERT luciferase reporter and 0.5μg preS2 expression plasmids were cotransfected into HepG2/BEL7402/LO2/COS-7.Meanwhile,different dose preS2 expression plasmids were used in HepG2.20ng SV40-Renilla was used as control to standardize the transcription efficiency.Cells were harvested 48h later in reporter lysis buffer.Luciferase assays were performed using the Dual-Luciferase Reporter Assay System according to the manufacturer's protocol.(5) HepG2 cells were transfected with pcS2-HA expression plasmids(preS2 gene fused with hemaglutinin tag) or pcDNA3.Western blot was performed to detect the expression change of c-myc.(6) To define the region of the hTERT promoter that was necessary for the preS2 response,series truncated/deleted/mutated hTERT promoter reporter plasmids were constructed.Luciferase assays were performed after cotransfected with pcS2-HA in HepG2 and COS-7.(7) In order to further explore if preS2 could bind with PRR,EMSA were involved with nuclear extract from both HepG2 cells transfected with pcS2-HA and freshly isolated HCC samples. 2.The regulation of Hepatitis B Virus and its viral proteins on ZHX2 expression in HCC(1) HBV and its viral proteins HBX/HBC/preS2 were overexpressed in HepG2 cells. Antisense RNA against HBX/ HBC/ preS2 were involved in HepG2.2.15. RT-PCR and Western blot were applied to detect the expression change of ZHX2.(2) 33 HCC samples were grouped by HBV infection status,RT-PCR was applied to detect the expression difference of ZHX2;HBV DNA loads were detected by real-time quantitative PCR with the HBV-positive HCC tissue genomic DNA, ZHX2 expression levels were analyzed with HBV DNA.(3) Total RNA of the liver tissues were extracted by TRIZOL from 2 HBV transgene mice and 3 normal BALB/c.All the mice were 6 months old.ZHX2 expression levels were determined by real-time quantitative PCR.3.The regulation of ZHX2 on GPC3 expression in HCC and the mechanism(1) RT-PCR was applied to detect the different expression levels of ZHX2 and GPC3 in HCC cell lines HepG2,Hep3B,SMMC7721 and immortalized liver cell line LO2.ZHX2 was overexpressed in cell lines express low ZHX2 levels.In contrast, siRNA-mediated ZHX2 knockdown was applied in cell lines express high ZHX2 levels.Then RT-PCR and Western blot were applied to detect the expression change of GPC3.(2) HepG2 cells were treated with 5-Aza-CdR of different dose to recover ZHX2 expression.RT-PCR and Western blot were applied to detect the expression change of GPC3.(3) 33 HCC samples were collected.ZHX2 and GPC3 expression were detected by Western blot.The relationship of ZHX2 and GPC3 expression was analyzed by Spearman analysis.(4) GPC3 core promoter reporter plasmid was constructed and named pGL3-GPC3-218.The promoter activity was validated in HepG2 and COS-7 by Dual-Luciferase Reporter Assay.Different dose of pcDNA3-ZHX2-HA(ZHX2 expression vector with HA tag) were cotransfected with pGL3-GPC3-218. pRL-TK plasmids were involved in each group to normalize transfection efficiency.Cells were harvested 48h later for luciferase assay.RESULTS1.The molecular mechanism for preS2-mediated transcriptional activation of hTERT in HCC development(1) In order to overcome the artificial effect of in vitro overexpression,preS2 antisense RNA were involved.RT-PCR and ELISA showed that preS2 antisense RNA could significantly decrease preS2 mRNA and HBsAg expression. Suppression of preS2 decreased hTERT expression.(2) 16 clinical tumor samples were involved and grouped by preS2 expression.As expected,much higher hTERT protein expression could be detected in preS2-positive HCC than that of preS2-negative tumors.(3) Using transfection of preS2 fused with EGFP tag,we verified the nuclei localization of preS2 which is consistent with previous study.This raises the possibility ofpreS2 as a transcriptional activator.(4) Results of cotransfection and luciferase assay clearly showed the preS2 mediated up-regulation on hTERT promoter activity in different cell lines,including immortal liver cell line LO2,HCC cell lines Be17402 and HepG2,and African green monkey kidney cell line COS-7.The up-regulation in HepG2 cells showed a preS2 dose-dependent manner.These data indicate that preS2 protein can transcriptionally upregulate hTERT via a non- liver-specific manner.(5) Recent evidence suggests that some virus-encoded proteins contribute to effect of telomerase through the c-myc binding sites.However,we failed to detect any change in c-myc expression after preS2 transfection,which indicated that c-myc play no obvious effect in the preS2-mediated upregulation of hTERT promoter.(6) To define the region of the hTERT promoter that was necessary for the preS2 response,a serial of hTERT promoter-reporter truncated or deletion constructs (pGL3B-895/pGL3B-371/pGL3B-306/pGL3B-349/pGL3B-329/pGL3B-318/ pGL3B-DELS2/ pGL3B-MUT371R/ pGL3B-MUT349S) were constructed. Cotransfection and luciferase assay demonstrate that preS2 upregulates hTERT promoter via a 65-bp region located in -371 to -307bp.Further assay identified a novel preS2 responsive-region(PRR) located between -349 and -329 bp upstream of the translational start site in the hTERT promoter.(7) EMSA verified the binding of preS2 to the PRR both in vitro and ex vivo.2.The regulation of Hepatitis B Virus and its viral proteins on ZHX2 expression in HCC(1) RT-PCR and Western blot results indicate that forced HBV and its viral proteins expression can reduce endogenous ZHX2 expression in SMMC7721.HBV and HBC showed stronger influence compared with HBX and preS2.Inversely, blocking HBV viral proteins expression in HepG2.2.15 can increase ZHX2 expression.Among all genes studies,HBC has the strongest effects.(2) 33 clinical tumor samples were involved and grouped by HBV infection status. Results showed that much lower ZHX2 mRNA expression could be detected in HBV-positive HCC than that of HBV-negative tumors.(p<0.01)(3) Studies in mice indicated that ZHX2 has a lower expression level in HBV transgenic mice than that in normal BABLC mice.(p<0.01)All above results support the hypothesis that HBV could regulate ZHX2 expression in HCC.3.The regulation of ZHX2 on GPC3 expression in HCC and the mechanism(1) RT-PCR results showed negative correlation between GPC3 and ZHX2 mRNA expression in different human cell lines.Using HepG2 and Hep3B,which express high GPC3 levels,we show that ZHX2 over-expression significantly decreases GPC3 secretion.Furthermore,using LO2 and SMMC7721 cells,which express low GPC3 levels,we use siRNA inhibition to show that GPC3 is de-repressed when ZHX2 levels are reduced.The inverse correlation combined with ZHX2 overexpression and siRNA experiment results indicate that ZHX2 could be responsible for the suppression of GPC3 in human liver cell lines.(2) 5-Aza-CdR pre-treated HepG2 cells showed an increased expression of ZHX2. Meanwhile,the expression of GPC3 decreased.This also represents the evidence that ZHX2 represses GPC3.(3) Spearman analysis of ZHX2 and GPC3 protein expression in 33 HCC samples showed an inverse correlation(r=-0.4968,p<0.01).(4) GPC3 promoter activity was validated in HepG2 and COS-7.Co-transfections of ZHX2 and GPC3-luciferase reporter genes demonstrate ZHX2 repression is governed by the GPC3 promoter.This represents the first direct evidence that ZHX2 represses GPC3.CONCLUSIONS1 Anti-sense blocking assay and clinical samples detection assay furtherly validated that HBV preS2 protein can transactivate hTERT.Cotransfection and Dual-luciferase assay proved that preS2 upregulates hTERT expression via the PRR element(-349～-329bp upstream the transcription initial site of hTERT gene) to involve HCC development.2 Overexpression and anti-sense blocking assay in cell lines,HBV transgene mice model and clinical samples detection assay illuminate that Hepatitis B Virus and its viral proteins suppress ZHX2 expression in HCC.3 Overexpression/siRNA assay in cell lines and clinical samples detection assay validated that ZHX2 is a repressor of GPC3 expression in HCC.GPC3 core promoter reporter plasmid was constructed successfully.Cotransfection and Dual-luciferase assay showed that ZHX2 effectively suppresses GPC3 promoter activity,which in further validates the suppression role of ZHX2 on GPC3.INNOVATIONS AND SIGNIFICANCES1 In this study we have found and defined a novel mechanism of viral liver carcinogenesis:HBV preS2 protein can function as a transactivator,interact with the PRR region of hTERT promoter to regulate telomerase activity,advance the development of HBV related HCC.2 We also proved that HBV and its viral proteins decreases ZHX2 expression in HCC.These results provide new insight into pathogenic mechanism of HBV related HCC.3 We have shown that ZHX2 represses AFP and GPC3 expression in human hepatocellular carcinoma.Since both AFP and GPC3 are frequently reactivated in liver tumors and ZHX2 appears to be silenced in some HCC samples,our studies may provide new insight into the development of HCC and may also provide new targets for drug development.