The Regulation Of The Hepatitis B Virus Core Protein On Human Telomerase Reverse Transcriptase Expression And Its Molecular Mechanism

Objectives:1. To study the regulation of hepatitis B virus core protein (HBc) on the expression of human telomerase reverse transcriptase (hTERT).2. To verify the key role of hTERT in the enhanced growth and proliferation of liver cancer cell mediated by HBc.3. To unravel the molecular mechanisms of HBc-induced hTERT transcription.Methods:1. The effects of HBc on the in vivo growth of HCC cellsOur previous results showed that HBc promoted liver cancer cell growth and clonal proliferation capacity in vitro. On this basis, we further verified this phenomenon in vivo with the nude mice subcutaneous tumor model. We subcutaneously transplanted1×107HepG2.2.15cells in0.2ml N.S in each mouse. Ten days later, the diameter of the tumor reached0.5cm. Then, the mice were randomly divided into two groups. Two microgram of pshRNA-HBc or pshRNA-NC was intratumorally injected respectively every three days. We measured the tumor size every two days for two weeks. Then the tumor growth curve was made. After two weeks, the nude mice were sacrificed and the tumor weight was measured. The mRNA expression of HBc and hTERT were detected with RT-PCR. 2. The regulatory effect of HBc on the hTERT expression2.1. The effect of HBc on hTERT protein expression in HCC cellsOur previous results showed that HBc promoted the hTERT mRNA expression in BEL7402cells, and pshRNA-HBc also inhibited hTERT mRNA expression in HepG2.2.15cells. On this basis, we transfected BEL7402. Hela or HepG2cells with pcDNA3-HBc-HA or pcDNA3.48hours later, the total protein was extracted and the protein expression of hTERT was measured via Western Blot. In order to define the expression and localization of hTERT, the immune-fluorescence assay was performed. Hela cells transfected with pcDNA3-HBc-HA or pcDNA3were labeled with hTERT antibody and stained with DAPI. Then the expression and localization of hTERT was checked by fluorescence microscopy.2.2. The correlation analysis between HBc and the expression of hTERT in HCC tissue samplesWe extracted RNA and total protein from16selected HCC tissue samples. According to the expression of HBc, the samples were divided into two groups, HBc positive and HBc negative. Western Blot was carried out to determine the protein expression of hTERT of the two groups. The protein expression intensity was determined via optical density scanning. Based on the results above, the scatter plot was obtained and the correlationship was analyzed.3. The role of hTERT in the growth of hepatoma cell promoted by HBcTo further understand the relationship between HBc, hTERT and cell growth, the BEL7402cell line were cotransfected with hTERT-si or NC-si and pcDNA3-HBc-HA or pcDNA3, the growth curve and colony formation assay was performed to determine the cell proliferation. On the other hand. HepG2.2.15cell was cotransfected with hTERT-si or NC-si and HBc shRNA plasmid, and the cell proliferation was examined to verify the role of hTERT in the growth of hepatoma cell promoted by HBc. 4. The regulatory mechanisms of the HBc on the expression of hTERT4.1. The regulation of HBc on the activity of hTERT core promoterIn order to clarify the mechanism by which HBc regulated the expression of hTERT, we transfected BEL7402, HepG2cell line with pcDNA3-HBc-HA or pcDNA3. pGL3B-hTERT371which carrying the hTERT core promoter reporter, and pRL-TK used as the internal reference plasmid.24h later, cells were lysed and dual luciferase fluorescence intensity was detected with96-chemiluminescence, and the results were analyzed statistically. On the other hand. HepG2.2.15cells were cotransfected with pshRNA-HBc or pshRNA-NC and pGL3B-hTERT371which carrying the hTERT core promoter reporter, using pRL-TK as an internal reference plasmid. The hTERT promoter activity was detected.4.2. To figure out the crucial promoter region of the hTERT promoter regulated by HBcTo further define the key region of the hTERT promoter that regulated by HBc, BEL7402and HepG2cell lines were transfected with pcDNA3-HBc-HA or pcDNA3and a series of truncated hTERT promoter reporter vector.24h later, the dual luciferase fluorescence intensity was detected by96-chemiluminescence. On the other hand, HepG2.2.15cell line was transfected with pshRNA-HBc or pshRNA-NC and a series of truncated hTERT promoter reporter plasmids. Then, the dual luciferase fluorescence intensity was detected by96-chemiluminescence.4.3. To determine the crucial site of the hTERT promoter regulated by HBc.Then, we referenced the relevant literature and analyzed the transcription factor binding sites in this crucial region of hTERT regulated by HBc, including the c-Ets-2binding site. According to the references, the transcription factor c-Ets-2played an important role in regulating and maintaining hTERT gene promoter activity. Therefore, we designed and constructed the hTERT core promoter reporter gene with mutated c-Ets-2binding site. Then BEL7402cells were transfected with pcDNA3-HBc-HA or pcDNA3and mutated or wild-type hTERT promoter reporter vector, using pRL-TK as an internal reference.24h later, the dual luciferase fluorescence intensity was detected with96-chemiluminescence. On the other hand. HepG2.2.15cells were cotransfected with pshRNA-HBc or pshRNA-NC and mutated or wild-type hTERT promoter reporter vector.24h later,96-chemiluminescence was applied to detect dual luciferase fluorescence intensity.5. The role of c-Ets-2protein and related signaling pathway in the hTERT expression regulated by HBc5.1. The effect of HBc on the c-Ets-2nuclear translocationAccording to literature reports, the roles of c-Ets-2protein in transcriptional regulation depend on its translocation from the cytoplasm to the nucleus. Therefore. HepG2cell lines were transfected with pcDNA3-HBc-HA or pcDNA3.24h later, we collected the nucleus and cytoplasm protein measured the expression pattern of c-Ets-2by Western Blot.5.2. The correlation analysis between HBc expression and nucleus location of c-Ets-2protein in the HCC samplesWe extracted the nuclear protein of HCC tissue samples, detected the cellular localization of c-Ets-2in HBc positive and HBc negative HCC sampleas by Western Blot respectively, and then analysed the protein expression intensity by optical density scanning. The statistical correlation analysis was carried out as well.5.3. The role of c-Ets-2in the regulation of hTERT expression by HBcTo further verify the critical role of c-Ets-2in regulating the expression of hTERT by HBc. HepG2cell lines was transfected with pcDNA3-HBc-HA or pcDNA3and c-Ets-2-si or NC-si.24h later, we detected the mRNA expression of hTERT by RT-PCR.5.4. The effect of HBc on ERK pathway.It was reported that the activation of the ERK signal pathway can phosphorylate c-Ets-2protein, thereby promoting its nuclear translocation process. Therefore. Hela and HepG2cell lines were transfected with pcDNA3-HBc-HA or pcDNA3. and the total protein was collected at24h,36h,48h respectively. And then we detected the expression of p-ERK1/2to verify whether HBc can affect the activation of the ERK signaling pathway.Results1. HBc protein promotes the growth of Hepatoma cell in vivoIn the nude mice experiment, we measured the tumor size and tumor weight. The results showed that the tumor volume and weight of pshRNA-HBc injected group was significantly smaller than those of pshRNA-NC injected group (P＜0.05). RT-PCR results showed that the hTERT mRNA expression of pshRNA-HBc group was significantly lower than that of control group.2. HBc upregulates the expression of hTERT2.1. HBc promotes the expression of hTERT protein in HCC cellsBEL7402. Hela and HepG2cell lines were transfected with pcDNA3-HBc-HA or pcDNA3. Western Blot showed that the expression of hTERT protein in HBc group was higher than that of pcDNA3group (P<0.05). Meanwhile, immunofluorescence showed that in Hela cells. hTERT protein was mainly localized in the nucleus and the overexpression of HBc promoted the expression of hTERT significantly.2.2. The expression of hTERT protein in HBc-positive HCC tumor was significantly increasedWe extracted RNA and protein from16selected HCC tissue samples and divided them into HBc-negtive group (6cases) and HBc-positive group (10cases) by RT-PCR. Western Blot showed higher hTERT protein expression in HBc-positive group than that of HBc-negative group. Statistical analysis also showed that the difference in the relative expression amount of hTERT/β-actin had statistical significance (P<0.05).3. hTERT played an important role in the promoted growth of liver cancer cells by HBcGrowth curve and colony formation showed that over-expression HBc can promote cell growth and colony formation ability (P<0.05) in BEL7402cell. However. cotransfection of hTERT-si almost abrogated this promoting effect (P>0.05). On the other hand. while knockdown of HBc can inhibit cell growth in HepG2.2.15cell and colony formation rate (P<0.05), cotransfection of hTERT-si eliminated the differences in cell growth and colony formation ability between pshRNA-HBc and pshRNA-NC group (P>0.05). These results suggested that HBc promotes cell growth and proliferation through hTERT.4. The regulatory mechanism of HBc on hTERT promoter activity4.1. HBc protein upregulated hTERT promoter activityCo-transfection and dual-fluorescence assay showed that in BEL7402cell. HBc dose-dependently upregulated the activity of hTERT core promoter (P＜0.001). HBc also promoted the hTERT core promoter activity in HepG2and COS-7cell lines (P <0.05). while pshRNA-HBc markedly decreased the hTERT core promoter activity in HepG2.2.15cell lines (P<0.05).4.2. HBc-responsive hTERT promoter region was located in-130～-197bpCo-transfection and dual-fluorescence results showed that in BEL7402and HepG2cells, pGL3B-hTERT371, pGL3B-hTERT306, pGL3B-hTERT197promoter activity were increased by HBc (P<0.05), while HBc had no significant effect on pGL3B-hTERT130promoter activity. And on the other hand, in HepG2.2.15cells. pGL3B-hTERT371, pGL3B-hTERT306, pGL3B-hTERT197promoter activity were decreased by pshRNA-HBc (P<0.05), while pGL3B-hTERT130promoter activity was not significantly altered by pshRNA-HBc. This showed that HBc-responsive hTERT promoter region was located in-130～-197bp. 4.3. The key sites for HBc transactivation is transcription factor c-Ets-2binding sitesRelated literature showed that there were several binding sites for transcription factors c-Ets-2. bHLH2, AP-2. NF-E2/AP2in-130～-197bp region. In view of the important role of transcription factor c-Ets-2in maintaining the transcriptional activity of hTERT, we constructed pGL3B-c-EtsMUT promoter reporter plasmid with the mutations in c-Ets-2binding sites. BEL7402cell lines was co-transfected with pGL3B-c-EtsMUT or wild-type pGL3B-hTERT371promoter reporter gene vector and pcDNA3-HBc-HA or pcDNA3. Co-transfection and dual-fluorescence results showed that HBc overexpression significantly increased wild-type pGL3B-hTERT371promoter activity (P<0.05). but had no effect on the activity of pGL3B-c-EtsMUT promoter. On the other hand. HepG2.2.15cell line was cotransfected with pGL3B-c-EtsMUT or wild-type pGL3B-hTERT373promoter reporter vector and pshRNA-HBc or pshRNA-NC. Dual fluorescence results showed that HBc knockdown significantly inhibited wild-type pGL3B-hTERT371promoter activity (P <0.05), but had no effect on the activity of pGL3B-c-EtsMUT promoter.5. c-Ets-2protein and related signaling pathway molecules played an important role in the regulation of hTERT expression by HBc5.1. HBc promoted the nuclear translocation of c-Ets-2protein in hepatoma cells.Western Blot showed that, in HBc-overexpressed HepG2cells, the expression of c-Ets-2protein in nuclear was significantly increased, while which in cytoplasm was reduced. This showed that HBc may promote the nuclear translocation of c-Ets-2protein.5.2. The expression of c-Ets-2protein in HBc-positive HCC tumor was slightly increasedWestern Blot detected the nuclear expression of c-Ets-2protein in HCC tissues. Results showed that the nuclear expression of c-Ets-2protein in HBc-positive HCC tumor was slightly increased than that in HBc-negtive HCC tumor. However, the statistical analysis showed that the difference had no statistical significance.5.3. c-Ets-2protein played an important role in the expression of hTERT mediated by HBcHepG2cell lines was transfected with pcDNA3-HBc-HA or pcDNA3and c-Ets-2-si. RT-PCR results showed that c-Ets-2siRNA markedly inhibited the expression of hTERT mRNA compared with the NC-si groups, and can eliminate the upregulating effect of HBc on hTERT expression.5.4. HBc promoted the activation of the ERK signaling pathway.Western Blot showed that in Hela cells, compared to the control group. pcDNA3-HBc promoted the phosphorylation of ERK1/2at36h,48h. and the expression of hTERT was also upregulated apparently. On the other hand, in HepG2cells. HBc also promote the expression of p-ERK1/2at24h. Combined with literature, the results suggested that the activation of the ERK pathway might be involved in the upregulation of hTERT expression and the nuclear translocation of c-Ets-2protein by HBc.Conclusions:1. HBc protein promotes the growth of liver cancer cells in vivo.2. HBc dose-dependently upregulates the expression of hTERT protein, and hTERT played an important role in the promoted growth of liver cancer cells induced by HBc.3. HBc upregulates hTERT promoter activity and the key sites for HBc transactivation is the binding sites of transcription factor c-Ets-2.4. c-Ets-2protein and related signaling pathway molecules played an important role in the upregulated effects of HBc on of hTERT expression.. Significances:1. For the first time, we revealed the regulation of HBc on the expression of hTERT. and proved the key role of the hTERT in the HBc regulation on the growth and proliferation of liver cancer cell. These results provided the direct expremental evidence for the involvement of HBc in the development of liver cancer.2. The intensive study of the molecular mechanism of the hTERT expression regulated by HBc was also conducted for the frist time. We had identified that the key sites for HBc transactivation is transcription factor c-Ets-2binding sites, and cleared that the nuclear translocation of c-Ets-2protein was crucial in the up-regulation of hTERT expression mediated by HBc. which provided new data for the pathway regulatd by HBc protein.