Effects Of Hepatitis B Virus Encoded Proteins On Human Telomerase Reverse Transcriptase Regulation Mechanism Of α-albumin Gene Expression

Hepatitis B virus(HBV) is regarded as a major etiological factor for acute or chronic hepatitis,liver cirrhosis and even hepatocellular carcinoma,which are worldwide health problems,especially in China.Clarifying the mechanism of HBV induced HCC will provide a powerful tool to prevent and control the development of HCC.Till now,the pathogenesis for HBV infection is still not clear.However,it is well known integrated HBV DNA might contribute to liver carcinogenesis.Studies found that HBV genome might integrate into the hTERT promoter region in HBV related HCC,in which the hTERT mRNA is transcribed from both the endogenous promoter and integrated HBV gene.Telomerase is multisubunit complex that forms the ends of eukaryotic chromosomes using its complementary RNA sequence as a template.Telomerase is generally inactive in normal somatic cells but is expressed in most human cancers and immortal cell lines.Recent reports support the concept that the activation of telomerase through transcriptional activation of the hTERT gene is another way by which human tumor-associated viruses can work.Integrated HBV DNA can encode two types of transcriptional activators,HBx protein and the PreS2 activators.Examination of the viral DNA sequences present in HCC has shown that sequences encoding for the hepatitis B virus X gene(HBx) and/or truncated envelope PreS2/S viral proteins are retained in a large proportion of tumor cells.These two proteins contribute the development of HBV related HCC. However,whether these two proteins may upregulate telomerase activity and the mechanisms are still unknown.Therefore,we firstly developed the study to explore the effects of HBx and PreS2 proteins on telomerase and further study the mechanisms.Objectives:1.To examine whether HBx and PreS2 protein regulate tolemerase activity through upregulation of hTERT transcription2.To map the HBx responsive element in the hTERT promoter3.To select a minimal region of HBx that is essential for activating hTERT promoterMethods1.Effect of HBx and PreS2 protein on telomerase and hTERT gene expression1.1 Effect of over-expression of HBx and PreS2 protein on telomerasepcDNA3-HBx or pcDNA3 were transfected into hepatoma cell line HepG2 by lipofectamine.After 48 h,the protein were extracted from the transfected cells and untransfected cells.Proteins were extracted from HepG2-preS2 cells which were stably transfected with preS2 gene.293 cells were used as a telomerase-positive control.293 cells were heat treated prior as telomerase-negative control.TRAP assay was done to detect telomerase activity.1.2 Effect of over-expression of HBx and PreS2 protein on hTERT mRNA expressionSemi-quantitative RT-PCR was used to detect hTERT mRNA expression in transfected and untransfected cells.1.3 Effects of PS-asODNs/HBx,PS-asODNs/preS2 on hTERT mRNA of HepG2.2.15 cellsPS-asODNs/HBx,PS-asODNs/preS2 which specifically targeted to HBx and preS2 were designed and synthesized together with random oligos,PS-rODNs. Different oligos were transfected into HepG2.2.15 cells by lipofectamine.After 48 h, hTERT mRNA was analysed by semi-quantitative PCR.2.Effect of HBx expression on hTERT promoter2.1 Constructions of a serial of reporter plasmids containing deleted and mutated hTERT promoterThe specific primers for deleted and mutated hTERT promoter were designed and synthesized for PCR amplification.The PCR products were cloned into pGL3-basic to construct reporter plasmids.The recombinants were assayed by restriction enzyme digestion,PCR and sequencing.2.2 Effect of HBx on hTERT promoter Cultured cells were transfected with pGL3B-TRTP and an increasing amount of pcDNA3-HBx.Luciferase assays were performed using the Dual-Luciferase Reporter Assay System,in which Renilla luciferase plasmids(pRL-tk) were cotransfected as a control to standardize the transcription efficiency.All experiments were performed at least three times in each plasmid and represented the relative luciferase activity as an average.2.3 Mapping of the HBx responsive element in the hTERT promoterA series of luciferase plasmids containing 5'-truncated hTERT promoter fragments were cotransfected with either a control expression plasmid(pcDNA3) or HBx expression vector(pcDNA3-HBx) into HepG2 cell line.The level of luciferase relative activity was determined 36h after transfection.2.4 Mapping of the HBx responsive key binding sites in hTERT promoterA series of luciferase plasmids containing point mutations in c-Myc or Spl binding sites of hTERT promoter were cotransfected with either a control expression plasmid(pcDNA3) or HBx expression vector(pcDNA3-HBX) into HepG2 cell line. The level of luciferase relative activity was determined 36h after transfection.2.5 Effects of HBx expression on Sp1 geneProtein and mRNA expression of Sp1,HBx andβ-actin in HepG2-pcDNA3,HepG2-HBx and HepG2 cells were detected by Western blot and semi-quantitative RT-PCR.3.A minimal region of HBx is essential for activating hTERT promoter3.1 Constructions of a serial of eukaryotic expression vectors containing truncated HBx geneThe specific primers for truncated HBx were designed and synthesized to amplify truncated HBx genes from pcDNA3-HBx.PCR products were cloned into pcDNA3 to construct serial of truncated HBx expression plasmids.The recombinants were assayed by restriction enzyme digestion,PCR and sequencing.3.2 Expression of truncated HBx gene in HepG2 cellsA Serial of truncated HBx vectors were transfected into hepatoma cell line HepG2 by lipofectamine.After 48 h,proteins were extracted from the transfected cells and untransfected cells.Western blot and RT-PCR were used to detect truncated HBx expression.3.3 Effect of truncated HBx on hTERT promoterCultured HepG2 were transfected with pGL3B-TRTP and a serial of truncated HBx.Luciferase assays were performed using the Dual-Luciferase Reporter Assay System,in which Renilla luciferase plasmids(pRL-tk) were cotransfected as a control plasmid to standardize the transcription efficiency.All experiments were performed at least three times in each plasmid and represented the relative luciferase activity as an average.Results1.HBx and PreS2 protein upregulate hTERT expression and increase telomerase activity in hepatoma cells1.1 Over-expression of HBx and PreS2 protein increase telomerase activityThe TRAP assay showed more DNA ladder appeared in HBx and PreS2 transfected cells,which indicated HBx and PreS2 protein increased telomerase activity.1.2 Over-expression of HBx and PreS2 protein upregulate hTERT mRNA expressionRT-PCR result revealed that hTERT mRNA expression in HBx or PreS2-transfected HepG2 cells was significantly higher than that of pcDNA3 transfected HepG2 cells.There is no significant difference in telomerase activity and hTERT mRNA expression between HepG2 and pcDNA3 transfected HepG2 cells.1.3 Blockade of HBx and PreS2 downregulate hTERT mRNA expressionRT-PCR confirmed that asODNs/HBx and asODNs/preS2 efficiently suppressed expression of HBx and PreS2 whereas the control rODNs had no affect on HBX and PreS2 expression.As expected,asODNs/HBX and asODNs/preS2 treatment decreased hTERT expression but had not effect onβ-actin expression.2.HBx regulates Sp1-mediated transcriptional activation of hTERT2.1 Construction of a serial of deleted and mutated hTERT promoter report vectorsA serial of deleted and mutated hTERT promoter genes were successfully amplified by PCR.The PCR products were cloned into pGL3-basic vector to construct recombinants.The recombinants were transformed into E.coli JM109 and the positive clones were selected by antibiotics,digestion,PCR and sequencing.2.2 HBx transactivate hTERT promoter significantlyDual-luciferase report assay indicated that HBx expression vector might transactivate hTERT promoter in HepG2 cells.The luciferase activity of the hTERT promoter was augmented along with the amounts of HBx.The luciferase activity was increased about 1.5-fold greater than the basal promoter activity.The HBx-mediated hTERT promoter activation was detected in other three cell lines SMMC-7721, Be17402 and COS-7 cells.2.3 The sequences between-197 and+37 in the hTERT promoter show HBx responsiveness.The luciferase activities of constructs pGL3B-TRTP,pGL3B-895,pGL3B-445, pGL3B-371,pGL3B-306 and pGL3B-197 showed 1.45,1.40,1.46,1.38,1.58 and 1.38-fold induction than basal pormoter,respectively,by HBx.Induction by HBx dropped to the basal level when deletion reached +37(pGL3B+37).All these support that-197 to +37 regions in hTERT promoter are essential for HBx-mediated hTERT activation.2.4 HBx activates the transcriptional activity of hTERT promoter not through c-Myc,but through Sp1Effects of HBx on various mutant constructs were initially examined in HepG2 cells.Dual-luciferase report assays showed that HBx up-regulated both wild-type promoter and engineered promoters with mutation in c-Myc binding sites.The results indicate that c-Myc binding sites are not involved in HBx-mediated activation of the hTERT promoter.We then mutated the five Sp1 sites within the promoter region of the construct pGL3B-del132.Dual luciferase report assay showed that the mutations introduced in Spl sites 2,4 and 5 decreased the induction of the hTERT promoter by HBx.Taken together,these data clearly demonstrate that HBx targets multiple Sp1 binding sites to induce the up-regulation of the hTERT promoter.2.5 HBx had no effects on the expression of Sp1 in HepG2Western blot and semi-quantitative RT-PCR showed that there were no significant differences in Spl gene expression between HBx transfected and control HepG2 cells(P＞0.05).3.The amino acids 50 to 140 of HBx were important for hTERT promoter activation3.1 Construction of a serial of HBx truncated eukaryotic expression vectorsA serial of truncated HBx genes were successfully amplified by PCR.The PCR products were digested and inserted into pcDNA3-HBx vector.The recombinants were transformed into E.coli DH5αand the positive clones were selected by antibiotics,digestion,PCR and sequencing.3.2 Truncated HBx expression in HepG2 cellsRT-PCR verified the mRNA expression of all truncated plasmids in HepG2 cells, however,western blot only showed the expression of TC-2,TC-3,HBX,TCN,TN-1 mutants and failed to prove the expression of small mutant,TC-1 and TN-2,probably because of the small size or/and short half-lives.3.3 The transactivator role of a serial of truncated HBx on hTERT promoterDual luciferase report assay showed that hTERT transcriptional activation was diminished in successive deletions of the N-terminal and C-terminal regions of the HBx-protein.In the N-terminal deletion mutant,removal of 50 amino acids did not have any effects on its ability to transactivate hTERT promoter in HepG2 cells. Larger,internal deletion extending from amino acids 1 to 97(TN-2) resulted in disappearance of transactivation.In the analysis of C-terminal deletion mutants, amino acids 140～154 could be deleted from HBx without affecting its ability to transactivate hTERT promoter.However,the HBx transactivating effect was lost in TC-1 and TC-2.Furthermore,removal of 50 amino acids of N-terminal deletion and 141～154 amino acids of C-terminal deletion(TCN) did not change its ability to transactivate hTERT promoter in HepG2 cells. Conclusion.1.HBx and PreS2 activator upregulate hTERT expression and increase telomerase activity.2.HBx stimulates hTERT promoter in a dose-dependent manner in different cells.3.The hTERT promoter sequences between-197 and+37 show HBx responsiveness in the hTERT promoter.4.HBx activates the transcriptional activity of hTERT promoter not through c-Myc, but through Sp1.5.The amino acids 50 to 140 of HBx are important for the transactivation of hTERT promoter.Our work provides new insights into mechanisms of HBV related HCC and also gives new potential targets for drug design against HBV related HCC. α-albumin(AFM, also called afamin) is a member of the albumin gene family that includes albumin (Alb), a-fetoprotein (AFP) and vitamin D-binding protein (DBP). Alb family proteins have significant structure similarities and are predicted to have evolved from an ancestral gene. The four genes have all been mapped to the 4q11-q22 region of chromosome and are specifically expressed in the liver. However, the four genes have specific patterns. Alb and AFP are highly induced in hepatoblasts and continue to be expressed at high levels in the fatal liver. Whereas Alb continues to be expressed in the adult liver, AFP is silence during the first several weeks after birth. However the repression is reversible as AFP can be reactivated in the adult liver in situations where there is renewed cell proliferation such as during liver regeneration or in hepatocellular cacinomas. DBP transcription begins mid-gestation in the fatal liver and continues to rise in the adult liver. AFM becomes activated at birth and continues to be highly expressed in the adult liver. Although mechanisms underlying the transcriptional control of the Alb, AFP and DBP have been studied by a number of laboratories, the basic for the tissue-specific and developmentally regulated expression of the AFM gene remains unexplored.Objectives1. To analysis the region of AFM promoter and study the promoter activity2. To find the transcription factor that regulate AFM promoterMethods1, Construct a serial of report plasmids containing mouse AFM gene promoterFull-length mouse AFM promoter sequence and deleted or mutated AFM promoter sequence were respectively cloned into pGL3-basic to construct report plasmids. Cotransfection and Dual-luciferase reporter assay were used to analysis the transcriptional activity of AFM promoter plamids in different cells (Hep3B, HepG2, HEK293 cells) .2. Regulation of HNF1 on AFM promoter An increasing amount of HNFl expression vector, pcDNA3-HNFla or pcDNA3-HNFlp was cotransfected into HEK293with pGL3-mAFM respectively cells. Luciferase assays were performed using the Dual-Luciferase Reporter Assay System, in which Renilla luciferase plasmids (pRL-CMV) were cotransfected as a control to standardize the transcription efficiency. All experiments were performed at least three times in each plasmid and represented the relative luciferase activity as an average.3. Regulation of HNFl on mutated AFM promoterHNFl expression vector (pcDNA3-HNFla or pcDNA3-HNFlp) was cotransfected into HEK293 cells with mutated AFM promoter plasmids. The level ofluciferase relative activity was determined 48h after transfection.4. Effect of HNFip on HNFla-induced activation of mAFM promoterDifferent amount of pcDNA3-HNFip together with O.lug pcDNA3-HNFlawere cotransfected with pGL3-mAFM into HEK293 cells. The level of luciferase relative activity was determined 48h after transfection.5. Analysis the binding of HNFl protein to HNFl binding sites on AFM promoterComputer analysis revealed two putative HNF-1 binding sites resided at -145 and -75 of the mAFM promoter, which was named sitel and site2. EMSA and gel supershift assay were involved to examine the actual binding of HNF-1 to the mAFM promoter. Nuclear extracts were prepared from Hep3B cells or HEK293 cells transfected with pcDNA3-HNFla or pcDNA3-HNFip. These extracts were incubated with radiolabeled DNA fragments containing the rat p-fibrinogen promoter HNFl site, sitel fragment, or site2 fragment.6. Regulation of C/EBP on AFM promoterC/EBP expression vector pcDNA3-C/EBPa or/and pcDNA3-C/EBPp was/were cotransfected into HEK293 cells with pGL3-mAFM report vector. The level of luciferase relative activity was determined 48h after transfection.Results1. A serial of report plasmids containing mouse AFM gene promoter were successfully constructedSequencing data of report vectors pGL3-mAFM and deleted or mutated AFM promoter vectors were correspondence with the design. The results of cotransfection and Dual luciferase assays demonstrated that pGL3-mAFM vector showed transcriptional activity in Hep3B and HepG2 cells. In constrast, HEK didn't confer demonstrable transcriptional activity. The findings suggest that AFM transcription is significantly activated in AFM positive cells.Transcription reduced in promoter activity was observed in constructs extending beyond position -158. However, luciferase activity was almost completely abolished (90% decrease in Hep3B cells) by a further deletion extending to postion -138. Similar results were obtained in mutation of HNF-1 site 1, which suppressed the AFM promoter activity about 90%. The abrogation of the HNF-1 site 2 by substitution of the nucleotide within this region (pGL3-mAFM) reduced transcription by 50% in Hep3B cells. The mutations of both HNF-1 sites completely eliminated the AFM promoter activity, suggesting that HNF-1 might be the most critical factor promoting initiation of AFM transcription.2. Mouse AFM promoter activity can be up-regulated by HNF-1The reporter plasmid pGL3-mAFM was co-transfected with increasing doses of pcDNA- HNF1αor 1βplasmid into HEK293 cells which is HNF-1 deficient cells. Normalized luciferase assay results clearly showed that HNF-1 upregulated mouse AFM promoter activity in a dose dependant manner in HEK293 cells, though the action of HNF1βwas only 10% of HNFla.3. The different activation of HNF1 on mutated mouse AFM promoter activityTo examine the site specificity of mAFM transcriptional activation by HNF-1, these mutated promoter reporter plasmids were cotransfected with HNF1αor 1βexpression plasmids in HEK293 cells. Cotransfection with mock vector was used as control. The mutation at the site lor site2 reduced HNF la-mediated activation to 50% or 40%. The mutation at the sitel didn't reduced HNF IB-mediated activation, while mutation at the site2 reduced HNF IB-mediated activation to 50%. All these suggest that HNF-1-mediated activation of mAFM promoter is primarily but not exclusively dependent on these two HNF-1 sites.4. The negative effect of HNF1βon HNF1α-induced activation of mAFM promoterpcDNA3-HNFlp and pcDNA3-HNFla were cotransfected into HEK293 cells with pGL3-mAFM. Dual luciferase assay results clearly showed that HNF1βdecreased HNF1α-mediated mAFM enhancement in a dose-dependent manner.5. HNF-1αand HNF-1βdirectly binds to the mAFM promoterEMS A was used to analyze the binding of HNF-1. HNF1 probed bands were detected in the extracts from HEK293 cells transfected with HNF1αor HNF1β, but not mock vector. These bands were also present in Hep3B cell extracts. The bands were completely competed by a homologous competitor, as well as by site 1 or site 2 oligos, but not by the site 1 or site 2 mutated oligos or unrelated oligos. The same results were observed with sitel and site2 probes. When supershift assays were carried, a supershift of specific shifted band was observed with the HNF1αantibody and HNF1βantibody, but not with NF-1 antibody in Hep3B extracts and HEK-293 cells extracts transfected with HNF1α/1βexpression vector.A modified competition binding study was carried out to extend the observation of the binding of HNF1 toβ-fibrinogen, sitel and site2. Competition for the P³²-β-fibrinogen complexes was carried out with 1-, 5-, and 10-fold molar excess of unlabeled p-fibrinogen, sitel and site2 oligos. Signal intensities of the residual bands were quantified. These data revealed that the relative affinities of each HNF1-binding site for HNFl bindings as follows: P-fibrinogen >sitel>site2。6. C/EBP don't regulate AFM promoter activityThe reporter plasmid pGL3-mAFM was co-transfected with pcDNA-C/EBPa or/and pcDNA-C/EBPp plasmid into Hep3B cells. Dual luciferase assay resultsclearly showed that C/EBP didn't alter mouse AFM promoter activity.Conclusion:1. Homologous analysis and reporter assay indicate that AFM promoter resideswithin the 336 bp region from transcriptional initiation site.2. Two HNF1 sites are found within the AFM promoter region and both HNFl sites appeared to constitute part of the mAFM promoter, but loss of the sitel site had a more profound effect than site2.3. HNF1αand HNF1βdirectly bind to the mAFM promoter and activate AFM promoter activity.4. HNF1αand HNF1βare important transcriptional factors for AFM promoter and HNF1βhas negative effect on HNF1α-induced activation of AFM promoter.5. C/EBP don't regulate AFM promoter activity.In this paper, we firstly studied the regulation mechanism of a-albumin gene expression. These results may help us to understand how to regulate liver-specific gene expression and provide new theory on mechanism of HCC development.