| HBV cannot be cleared entirely because HBV DNA often integrating into host DNA result in persistent virus infection,which has become a major worldwide health problem.Till now,the pathogenesis for HBV infection and HCC development are still not clear.But studies showed that imbalanced apoptosis of hepatocytes induced by HBV infection play an important,even decisive role in liver injury,the prognosis and regress for patients with HBV infection.TRAIL,TNF related apoptosis inducing ligand,was identified and named after the FasL,is a new member of the TNF superfamily.The distinguished characteristic of TRAIL from other members of TNF superfamily is that TRAIL selectively induces apoptosis of tumor cells or virus-infected ceils while has no cytoxicity on normal tissues.Recent studies demonstrate that TRAIL play important roles in clearance of CMV,ECMV and many other virus infection.But the mechanism of TRAIL effecting on HBV infection and HCC development is not clear.There're at least four open reading frames(ORF)in HBV genome,which code corresponding viral proteins.Then,which are the most critical fragment(s)for effects of HBV on TRAIL-induced apoptosis? What key points do these fragments affect in the TRAIL signaling pathway? Therefore,on the basis of these research backgrounds, we firstly demonstrated that HBV genome and HBx protein sensitize hepatocytes to TRAIL-induced apoptosis through upregulating Bax.But what is the key molecular modulated by MHBs(t)for sensitizing to TRAIL-induced apoptosis? And if another important protein,HBc also affects TRAIL-induced apoptosis? On the basis of these research backgrounds,we firstly study the effect of HBc on TRAIL-induced hepatocyte apoptosis and the molecular mechanisms of MHBs(t)and HBc.OBJECTIVES:1.To demonstrate the effect of HBc on TRAIL-induced hepatocyte apoptosis and to probe the mechanism of HBc on TRAIL-induced hepatocyte apoptosis.2.To determine the critical molecules which participate in upregulating TRAIL-induced apoptosis by MHBs(t).METHODS:1.1 Construction of eukaryotic expression vectors containing HBc geneThe specific primers for HBc were designed and synthesized.HBc gene was amplified from pUC19/3HBV by PCR.PCR products were digested and inserted oriently into pcDNA3.0.The recombinants were transformed into E.coli DH5αand the positive clones were selected by antibiotics,restriction enzyme digestion,PCR and sequencing and named pcDNA-HBc.1.2 Establishment of hepatoma cell line stably-expressing HBcpcDNA-HBc or pcDNA3.0 was transfected into hepatoma cell line BEL7402 and SMMC7721 mediated by lipofectamine.The positive cell clones were obtained by selection with G418 and named as BEL7402-HBc,BEL7402-pcDNA3 and SMMC7721-HBc,SMMC7721-pcDNA3 cells respectively.Semi-quantitative RT-PCR was used to detect the mRNA expression of HBc;Western blot was used for detection of HBc protein.1.3 Expression of HBc in the hepatocytes of mice by hydrodynamic injectionBALB/c mice were transfected by hydrodynamics-based tail vein injection. Groups of mice were injected with pcDNA3.0,pcDNA3-HBc,pcDNA3-HBV1.1,and pcDNA3-HBV1.1+pcDNA3-HBc,respectively.48h later,mice were sacrificed and the HBc expression was detected by immunohistochemical staining.HE staining and serum ALT levels were also detected to observe the liver injury.1.4 Effects of HBc on TRAIL-induced apoptosis Hepatoma cell model: BEL7402,BEL7402-pcDNA,BEL7402-HBc;SMMC7721,SMMC7721-pcDNA3, SMMC7721-HBc were respectively exposed to different concentrations of TRAIL.24h later,the growth inhibitory rates for each group were detected by CCK-8 in order to select the optimal concentration of TRAIL.Then all groups of cells were treated with the optimal concentration of TRAIL and the apoptosis rates were detected by TUNEL.A 16-mer PS-ODNs,complementary to HBc poly-A region of HBV genome(adr subtype),called PS-asODNs/HBc,was synthesized.BEL7402-HBV1.1 cells,pretreated with PS-asODNs/HBc or PS-rODNs for 24h,were exposed to 10ng/mL TRAIL for another 24h and then the apoptosis rates were detected by TUNEL.Additionally,BEL7402-HBx cells transfected with HBc for 24h,were exposed to 10ng/mL TRAIL for another 24h.TUNEL assay was used to examine the apoptotic rate.Mouse model:BALB/c mice were transfected by hydrodynamics-based tail vein injection. Groups of mice were injected with pcDNA3.0,pcDNA3-HBc,pcDNA3-HBV1.1,and pcDNA3-HBV1.1+pcDNA3-HBc respectively.48h later,the paraffin sections were stained using TUNEL kit and observed on fluorescence microscope.Simultaneously, liver tissues grinded into single cell suspension were labeled by TUNEL and apoptotic rates were detected by flow cytometry.2.Molecular mechanisms of HBc expression on TRAIL-induced apoptosis1)Extracellular level:Hepatoma cell model:mRNA expression level of TRAIL receptors on BEL7402,BEL7402-pcDNA3,BEL7402-HBc;SMMC7721,SMMC7721-pcDNA3,SMMC7721-HBc cells were detected by semi-quantitative RT-PCR;protein expression level by Western blot and flow cytometry.Mouse model:BALB/c mice were transfected by hydrodynamics-based tail vein injection.Groups of mice were injected with pcDNA3.0,pcDNA3-HBc, pcDNA3-HBV1.1,and pcDNA3-HBV1.1+pcDNA3-HBc respectively.48h later, mice were sacrificed and DR5 expression was detected by immunohistochemical staining,flow cytometry and Western blot. Clinical samples:Sera of 110 patients with chronic hepatitis B were collected.HBc expression and soluble DR5 levels in sera were detected by ELISA kit.Liver samples from 32 chronic hepatitis B patients were obtained by liver centesis.DR4,DR5 expression was determined by immunohistochemistry as described above.2)Intracellular level:Hepatoma cell model:BELT402,BEL7402-pcDNA3,BEL7402-HBc cell were treated with 10ng/mL TRAIL.Twenty-four hours later,expressions of procaspases 3, 8,9 and Bid were detected by Western blot.The expression levels of Bax and FLIP were detected by RT-PCR and Western blot.Mouse model:BALB/c mice were transfected by hydrodynamics-based tail vein injection.Groups of mice were injected with pcDNA3.0,pcDNA3-HBc, pcDNA3-HBV1.1,and pcDNA3-HBV1.1+pcDNA3-HBc respectively. Fourty-eight hours later,mice were sacrificed and expressions of procaspase 3,8,9 and Bid were detected by Western blot.3)The effect of HBc on DR5 promoterBEL7402-pcDNA3 and BEL7402-HBc cells were transiently transfected with pDRSPF.48 h later,the cells were harvested and the luciferase assay was used to detect the luciferase activity.3.Microarray analyses after HBc transfection in BELT402 cellsThe total liver RNA was extracted and hybridezed on an oligonucleotide chip containing 20,000 genes.Significant differentially expressed genes were analysed by bioinformatics approach.4.The molecular mechnisms of MHBs(t)upregulating TRAIL-induced apoptosis in hepatoma cellsActivations of ERK2 were detected by Western blot in BEL7402,BEL7402-pcDNA3 and BEL7402-MHBs(t)cells.BEL7402-cDNA3 or BEL7402-MHBs(t),pretreated with PD98059,was exposed to10ng/ml TRAIL. Twenty four hours later,TUNEL flowcytometry was used to detect the apoptosis rate and western blot was used to detect activations of procaspase 3,8,9. RESULTS1.HBc downregulates TRAIL-induced apoptosis1.1 Construction of eukaryotic expression vector pcDNA-HBcHBc gene was successfully amplified by PCR.The PCR products were digested and ligated to construct the recombinants.The recombinants were transformed into E.coli DH5αand the positive clones were selected by antibiotics,digestion and sequencing,and named pcDNA-HBc.1.2 Establishment of hepatoma cell lines stably-expressing HBcBEL7402 and SMMC7721 cells were transfected with pcDNA-HBc or pcDNA3.0 and selected with G418.The stable cell clones were termed as BEL7402-HBc,BEL7402-pcDNA3 and SMMC7721-HBc,SMMC7721-pcDNA3 respectively.HBc expression can be detected using RT-PCR and western blot.1.3 Expression of HBc in the hepatocytes of mice by hydrodynamic injectionBALB/c mice were transfected by hydrodynamics-based tail vein injection. Groups of mice were injected with pcDNA3.0,pcDNA3-HBc,pcDNA3-HBV1.1,and pcDNA3-HBV1.1+pcDNA3-HBc,respectively.48h later,mice were sacrificed and the HBc expression can be detected by immunohistochemical staining.HE staining and serum ALT levels demonstrated that compared with pcDNA3-HBV1.1 group,pcDNA-HBV1.1+pcDNA-HBc group had less liver injury(P＜0.01).1.4 HBc expression downregulates TRAIL-induced apoptosis Hepatoma cell model:CCK-8 assay showed that,treated with the same concentration of TRAIL, BEL7402-HBc,SMMC7721-HBc cells had lower cytotoxicity rates than control cells. The difference was most dramatic when the concentration of TRAIL was 10ng/mL. Then this concentration was determined as the optimal one.Results of TUNEL displayed that,treated with 10ng/mL and 100ng/mL TRAIL respectively, BEL7402-HBc,SMMC7721-HBc cells had lower apoptisis rates than control cells(P＜0.01).BEL7402-HBV1.1 cells,pretreated with PS-asODNs/HBc or PS-rODNs for 24h, were exposed to 10ng/mL TRAIL for another 24h.The apoptosis rates displayed HBc knockdown significantly up-regulated the sensitivity of BEL7402 cells transfected with pcDNA3-HBV1.1 to TRAIL-induced apoptosis(P＜0.01).Additionally, BEL7402-HBx cells with HBc significantly inhibited TRAIL-induced apoptosis(P＜0.01).Mouse model:TUNEL in situ displayed that pcDNA-HBV1.1 group had higher lower apoptosis rate than pcDNA-HBV1.1+pcDNA-HBc.Results of flowcytometry displayed compared with pcDNA3-HBV1.1 group,pcDNA-HBV1.1+pcDNA-HBc group had lower apoptosis rate(P＜0.01).2.Mechanisms of downregulation of TRAIL-induced apoptosis by HBc1)Extracellular level:Hepatoma cell model:Semi-quantitative RT-PCR,real-time PCR,Western blot and flow cytometry showed that DR5 expression was dramatically decreased compared with control cells(P＜0.01),but the other TRAIL receptors expression had no significant differences(P＞0.05).Mouse model:Immunohistochemical staining,flow cytometry and Western blot showed that DR5 expression was decreased in pcDNA-HBc group and pcDNA-HBV1.1+pcDNA-HBc,compared with pcDNA3.0 and pcDNA-HBV1.1, respectively(P＜0.01).Clinical samples:Sera of 110 patients with chronic hepatitis B were collected. The result showed that 81%of chronic hepatitis B patients were HBc-positive in serum.Soluble DR5 in patients' sera was also decreased compared with nomal control(p＜0.001).DR5 but not DR4 expression was significantly reduced in both intensity and the expressing cell number in chronic hepatitis B patients.Intracellular level:Hepatoma cell model:Treated with 10ng/mL TRAIL,BEL7402-HBc cells had lower levels of procaspase 3,8,9 and Bid activation,compared with control cells(P＜0.01).But there were no differences in the expression of FLIP and Bax(P＞0.05). Mouse model:Western blot showed that the activation of procaspases 3,8,9 and Bid were lower in pcDNA-HBV1.1+pcDNA-HBc group,compared with pcDNA-HBV1.1 group.3)HBe down-regulates the DR5 promoter activityLuciferase assay showed that BEL7402 cells transfected with control plasmid pcDNA3 had a high DR5 promoter activity;this was significantly decreased in BELT402 cells transfected with HBc(P＜0.01).3.Microarray analyses after HBc transfection in BEL7402 cellsGene chip showed that DR5 expression in BEL7402-HBc was dramatically decreased compared with BEL7402-pCDNA3 cells.Additonally,35%of the differentially expressed genes were involved with carcinogenesis.10%of the differentially expressed genes were involved with hepatocellar carcinogenesis. Changes of 4 HCC-related gene(Sulf-2,ATIP1,LOXL1,DLC-1)has been verified by Real-time PCR and obtained the same result with gene chip.4.The molecular mechnisms of MHBs(t)npregulating TRAIL-induced apoptosis in hepatoma cells4.1 MHBs(t)enhances the activation of ERK2Western blot was used to detect the activation of ERK2.The result showed that,in BEL7402 cells stably transfected with MHBs(t)but not control BEL7402 cells,ERK2 was significantly activated.4.2 ERK2 activation is necessary for MHBs(t)-mediated sensitization to TRAILBEL7402-cDNA3 or BEL7402-MHBs(t),pretreated with PD98059,was exposed to10ng/ml TRAIL.The result showed that PD98059 decreased the apoptosis rate induced by TRAIL in BEL7402-MHBs(t),but not in control cells.These data indicate that ERK2 is required for MHBs(t)-mediated sensitization to TRAIL.4.3 ERK inhibition blocks TRAIL-induced degradation of procaspases-3 and 9Western blot was used to detect the activation of procaspases-3,8,9.The result demonstrated ERK inhibition PD98059 remarkably blocked the degradation of procaspases-3 and 9 in BEL7402-MHBs(t)cells,no effects on procaspase degradations were observed in BEL7402-pcDNA3 cells.The results further demonstrate that ERK2 is required for MHBs(t)-mediated sensitization to TRAIL.CONCLUSIONS:1.HBc decreases the sensitivity of hepatoma cells towards TRAIL.2.HBc has no effects on the expression of FLIP and Bax in BEL7402 cells but down-regulates the activation of procaspases 3,8,9 and Bid.Importantly,HBc decreases the expression of DR5.The further mechanism showed that HBc decreases the DR5 promoter activity.3.HBc expression may involved with hepatocellar carcinogenesis.4.ERK2 activation is necessary for MHBs(t)-mediated sensitization to TRAIL. Additionally,ERK inhibition blocks TRAIL-induced degradation of procaspases-3 and 9.INNOVATIONS AND SIGNIFICANCES:1.Through establishment of stable hepatoma cell models and blockade by phosphorothioated antisense oligonucleotides,the present study demonstrates that the effects of HBc on TRAIL-induced apoptosis and firstly reports the downregulation of TRAIL-induced apoptosis by HBc.2.From extracellular level,intracellular level and mitochondrial-dependent, -independent pathway,the present study systematically probes into the molecular mechanisms for effects of HBc on TRAIL-induced apoptosis and proposes that HBc influences TRAIL-induced apoptosis by regulating both mitochondria-dependent pathway and mitochondria-independent pathway.3.By application of hydrodynamic injection,HBc expression in hepatocytes of mice further confirmes that HBc downregulates TRAIL-induced apoptosis, which establishes a novel experiment technique for functional study of HBc in vivo.4.It is the first time to prove that HBc decreases DR5 promoter activity,which is a basic for further mechanism study.5.Through establishment of stable hepatoma cell models,the present study firstly demonstrates that ERK2 activation is necessary for MHBs(t)-mediated sensitization to TRAIL.
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