The Key Control Of Hbv-related Hepatocellular Carcinoma (hcc), Designated Closed Tumorigenesis

Primary hepatocellular carcinoma (HCC) is one of the most comman malignant tumors in China. At present, because of no effective treatments, patients with HCC have very poor prognosis. With the development of the theory and techniques of molecular biology, gene therapy is becoming an important component of HCC comprehensive treatments. Anti-sense nucleotide technique which can block and inhibite the gene expression at molecular level, is becoming the focus of gene therapy. It can be used in the regions of anti-virus and anti-tumor.It is demonstrated that the infection of HBV and its integration into chromosome of hepatocytes, and the following activation of telomerase, the activation of oncogenes or inactivation of tumor-suppressor genes, were the key factors during the hepatocarcinogenesis.There was a very close relationship between telomerase and cell immortalization or caicinogenesis. Human telomerase catalytic subunit (hTERT), an important component of telomerase, is very necessary for the activation of telomerase in the tumor and immortalized cells. Researches showed that the positive rate of telomerase was 85% in HCC tissues. Among them, HBV positive patients showed telomerase 100% positive. It is demonstrated that in hTERT promoter region, there was a integration site of 65bp sequence of HBVpreS2. This result suggested that the integration of HBVpre2 resulted in the activation of telomerase, and followed with the cells malignant transformation and the occurrence of HBV related HCC. Therefore, the integration site of HBVpreS2 in hTERT promoter region was one of key sites for the carcinogenesis of HBV related HCC.The regulation to hTERT directly affects the activity of telomerase. At the 5 ' flanking region of hTERT promoter, there are many cis-acting elements which regulate hTERT activity, e.g. SP1 binding site, AP-2 (activator protein-2) binding site, Ebox (binding site of transcription factor bHLHZ family, e.g. c-Myc), etc. Oncogene c-Myc can promote the expression of hTERT through binding with Ebox. Becausec-Myc was the target of varies of carcinogenic factors, e.g. mutant p53 protein, viral oncoprotein, it was supposed that these factors activate c-Myc gene, and followed with promotion of hTERT expression and the activation of telomerase, resulting the occurrence of tumors. Therefore, the activation of hTERT and c-Myc binding site in hTERT regulation region was another key site for hepatocarcinogenesis.OBJECTIVEAt first, anti-sense oligonucleotides (asON) complementary to c-Myc binding site was synthesized and was added to HepG2.2.15 cells, a hepatic tumor cell line with the integration of HBV genome, to study the inhibition effect on the malignant phenotype of tumor cells. Then, using the AdEasy-1 system as gene transfer vector, anti-sense RNA adenoviral vectors complementary to the key sites of HBVpreS2 integration site, c-Myc binding site and hTERT promoter region were constructed. After the package of 293 cells and amplification, quantities of high purified recombinant adenoviral particles were obtained, which could specifically block the key sites for the onset of HBV related HCC. HepG2.2.15 cells were infected with these particles. The inhibition effect of anti-sense RNA on the growth of hepatoma cells were discussed in vitro and in vivo. The aim of this study was to establish a foundation for HBV related HCC gene therapy.METHODSSection 1: Study on the inhibition effect of asON complementary to c-Myc binding site (E-box) in telomerase hTERT promoter to hepatoma cellsThe nucleotides of -192-----178 site (containing E-boxes) in hTERT promoterregion were selected as target sequence. The P-modified asON was synthesized and added to HepG2.2.15 cells at the concentration of 10|xmol/L. Using the methods of PCR-ELISA and MTT, the activity of telomerase and the inhibition effect on growth of hepatoma cells were detected respectively. A hepatoma mouse model was obtained after injected with 5xlO6 HepG2.2.15 cells. The inhibition effects of as-hTERT on the growth of human hepatoma cells were studied through three different administration of asON in vivo.Section 2: Study on the inhibition effect of anti-sense RNA to the growth of hepatoma cells through specific blockage of key sites of hepatocarcinogenesis.2.1 To construct sense/anti-sense RNA expression vectors complementary to key sites using AdEasy-1 system.According to the characteristics of gene structure of telomerase hTERT promoterregion, PCR primers were designed to amplify gene fragments of HBVpreS2 integration site, c-Myc binding site and the complete hTERT promoter region. Digested by restriction endonuclease Kpn I, the gene fragments and adenoviral shuttle vectors—- pShuttle-CMV\ pAdTrack-CMV were ligated together to construct sense/anti-sense shuttle vectors, identified with restriction endonuclease digestion, PCR amplification and DNA sequence analysis. The sense/anti-sense shuttle vectors linerized by the digestion of Pme I were cotransformed into BJ5183 bacteria with adenoviral backbone plasmid (pAdEasy-1 plasmid) together to perform homologous recombination. In this way, sense/anti-sense RNA adenoviral genome vectors complementary to key gene fragments— HBVpreS2 integration site, c-Myc binding site, active region of hTERT promoter, were obtained through LB plate selection with 25 y g/mL kanamycin and identification with digestion of Pac I or BamH I..2.2 Amplification and assay for titration of rAdSense/anti-sense RNA adenoviral genome vectors digested by restriction endonuclease Pac I were transformed into HEK 293 cells mediated by Lipofectamine?. The cells were harvested when a visible cytopathic effect was observed (about 7-10 days postransfection), and lysised through freeze/thaw/vortex for four cycles, then the supernatant was gathered by centrifuge at 4 °C. Transfer the supernatant to another flask of 293 cells to amplify. Therefore, crude extracts of rAd particles were obtained and purified through CsCl gradient centrifugation. The titration of virus was detected with plaque forming test or FCM.2.3 Study of the growth inhibition effect on HepG2.2.15 cells in vitroHepG2.2.15 cells were cultured in 24-well plate at the concentration of 2xlO5/ml. 24 hr later, the culture medium was discarded, and the cells were infected with virus of 40 multiplicity of infection (MOI). Then, the cells apoptosis was detected throughFCM, apoptosis bodies were observed with transmission electromicroscope. Telomerase protein expression and activity was detected by the methods of RT-PCR^ PCR-ELISA respectively. The cytopathic effect was observed with Laser Confocol Microscopy, and the HBV antigen expression was detected with ELISA. Thus, inhibition effects on HBV related hepatoma cells of anti-sense RNA specific blockingthe key sites were studied.2.4 Studies in vivoThere were two methods to study the inhibition effects of antisense RNA on nu/mice models. After injection with 5xlO6HepG2.2.15 cells, the mice with tumor diameter >=0.5 cm were selected for the following test: the animal was injected intra-tumor with 1010 MOI anti-sense virus, once a week for four times, then the inhibition effect on tumor growth was observed. In another team, mice models were given 1010 MOI virus 72 hr postinjection with HepG2.2.15 cells. At the same time, cell control and NS control were installed.2.5 Effect on HBV antigen expressionHepG2.2.15 cells were incubated in a 96-well plate, and infected with recombinant adenovirus at 40 MOI and incubated for another 72 hr. Then the supernatant was collected to detect the quantity of HBsAgN HBeAg with ELISA, and the the inhibition ratio of HBV antigen expression was calculated.2.6 Cooperaction of two types of antisense-RNAAdenovirus-pAd-ashmyc infected HepG2-asmyc cells, a cell line which was transformed with an antisense-RNA expression vector complementary to the exon of c-Myc gene. 2days postinfection, telomerase activity and apoptosis were detected through PCR-TRAP-ELISA and Annexin V-FITC/PI staining respectively.Adenovirus-pAd-ashS2 infected HepG2.2.15-as-preS2 cells, a cell line which was transformed with an antisense-RNA expression vector complementary to the HBV preS2 gene. 2days postinfection, telomerase activity and apoptosis were detected through PCR-TRAP-ELISA and Annexin V-FITC/PI staining respectively.RESULTS1. Antisense olignucleotides targeting at the binding-site of c-myc in hTERT promoter region strongly inhibited malignant behavior of HepG2.215 cell lines.HepG2.215 cells were incubated with ashTERT at concentration of 10 u mol/L, not only telomerase activity(A450nm) dramatically decreased compared with HepG2.215 control cells(p<0.05), but also the growth of the HepG2.215 cells was inhibited.The mice were treated with three kinds of drug administrations when tumors were formed after injected with 5 X107 HepG2.215 cells: continuously injection into tumor, complex injection and injection once. All the three kinds of methods could effectively inhibit the growth of tumor cells. Apoptosis was observed in the first method, while the strong growth inhibitory effects were observed in another two methods. The results demonstrated that as-hTERT drugs could effectively arrest the growth of tumor cells. Small dose and superadditional drug-administration at early period could get a perfect effect in tumor therapy.The results revealed that E-box region was a workable targeting gene to be blocked with antisense olignuleotides in tumor gene therapy. Because of the coherence of human telomerase structure, inhibiting the activity of telomerase with asON was the perfect targeting site to kill tumor cells. This work has been published and embodied by SCI.2.Successfully construct sense/antisense RNA expression vetors targeting at the key sites of hepatocarcinogenesisTo resolve the problem of expensive cost and using repeat of artificial antisense olignuleotides, in this study, the antisense RNA eukarycyte expression vectors complementary to the key sites, such as HBVpreS2 integrated site, cmyc-binding site and hTERT promoter region, were successfully constructed.The gene fragments of HBVpreS2 integrated site, cmyc-binding site and hTERT promoter region were amplified through PCR, these gene fragments were 251bp, 370bp and 1.8kb respectively. Fragment of HBVpreS2 integrated site was cloned intopAdTrack-CMV and identified by digestion with Kpn I, PCR amplification and sequencing analysis. C-Myc binding site and hTERT overall promoter region were cloned into pShuttle-CMV vector. Then the sense/antisense shuttle vectors of pShuttle-s/ashmyc, pShuttle-s/ashTERT and pShuttle-shTERT were obtained. After homologous recombination in BJ5183, positive clones were selected out by kanamycin plates and digested with Bamh I and Pac I . Then sense/antisense advenoviral genome vetors were obtained.3. Obtaination of high titration recombinant advenovirusSense /antisense advenoviral genome vetors were transformed into HEK293 cells mediated by lipofectamine. Cells were harvested to freeze and lysis repeatly. 293 cells were infected with supernatant, then recombinant virus were amplified. After CsCl-binding purifation, the rAd with high titration were obtained. With plaque forming test, the titers of pAd-a shS2. pAd-ashmyc^ pAd-ashTERT were 2.1 X1011 pfu, 2.8 X1011 pfu, 1.6 X10n pfu respectively. While the titers of pAd-ashS2, pAd-TracK were 2.2X 1011 pfu, 1.7X 10n pfu respectively by flow cytometry.AdEasy-1 system is a new kind of deficient adenoviral expression vector system. The shuttle vectors have CMV promoters. And so the interested gene can be expressed efficiently. The shuttle vector pAdTracK-CMV has 2 CMV promoters. Downstream of one is MCS where interested gene can be inserted, while the other has GFP gene, whose protein can be used to detect the genetic expression and the viral titer. Homogeneous recombinant between shuttle vector and backbone plasmid can be done convenientely in BJ5183 cells. Because of the higher efficiency of transformation and stable propagation in bacteria, adenoviral vector is a ideal vector system for gene therapy.4. Inhibitory effect on hepatoma cells growth in vitro4.1 Targeting at the key site of carcinogenesis could enhance apoptosis effect on hepatoma cellsHepG2.2.15 cells were infected with antisense RNA of adenovirus(PAd-ashS2, PAd-ashmyc, PAd-ashTERT) at 40 MOI. 2d or 4d poatinfection, the cells were harvested to detect apoptosis. The result was that antisense RNA targeting at the keysite of HBVpreS2 integration site could reduced s-stage cells and the peaks of apoptosis of tumor cells were 12.2%, 23.1% respectively. Dying with Annexin V-FITC/PI, cells treated with pAd-ashmyc and PAd-ashTERT were detected apoptosis with flow cytometry. The peaks of apoptosis were 40.7%, 37.5%(2d) and 3.4%(80% cell death), 8.4%(50.8% cell death) respectively. Furthermore, apoptosis bodies were observed by electronic microscope. While the apoptosis peaks in HepG2.215 cells control, vector control and sense control were 2.7%, 1.7%, 1.3%(2d) and 3.3%, 6.8%, 0.9%(4d) respectively. There was a significant difference between antisense groups and control groups(p<0.05).4.2 Targeting at the key site of carcinogenesis could effectively inhibit hepatoma cells growthCell growth inhibition was detected by the method of MTT. After infected with rAd, the cells grew very slowly, and cytopathic effect was observed: cytoplasm shrunk to round and cells congregated together, falling off from culture bottles. While vector controls and sense-control cell growth wass perfect and culture bottles were full of cells 48h later.4.3 Targeting at the key site of carcinogenesis could effectively decrease the telomerase activity of hepatoma cellsHepG2.2.15 cells were infected with antisense rAd. The cells were harvested to abstract RNA, and telomerase protein and activity were detected through RT-PCR and TRAP-PCR-ELISA respectively. The results was that the telomerase activity in pAd-ashS2, pAd-ashmyc, pAd-ashTERT treated cells were 1.175, 2.8, 2.69 respectively, contrast to cell control, vector control and sense control 4.2, 4.013, 4.315 respectively. There were significant differences between antisense groups and control groups, but there were not significant differences among 3 kinds of antisense groups (P>0.05).4.4 Targeting at the key site of carcinogenesis could effectively inhibit HBV antigen expression.The supernatant of HepG2.2.15 cells infected with rAd and the serums of mice models were harvested for detecting HBsAg, HBeAg. The inhibitory rate of HBsAg,HBeAg were 32.5%, 38.3% respectively.The results demonstrated that antisenseadvenovirus could affect HBV antigen expression.4.5 Antisense advenovirus could efficiently infect hepatoma cell in vitroOne day postinfection with pAd-ashS2, GFP expression in HepG2.2.15 cells was observed under fluorescence microscopy, and the density of fluoresence intensified along with time. Strong fluorenscence and CPE were observed with Laser Confocol Microscopy. GFP expression rates of rAd infected cells and HepG2.2.15 control were 67% and 1.0% respectively through flow cytometry.The results in vitro study demonstrated that antisense advenovirus could efficiently infect hepatoma cells and block the key sites during hepatocarcinogenesis. Not only tumor cell apoptosis was induced, but also the cells growth and telomerase activity were inhibited. Therefore, the hepatoma cells malignant behavior were inhibited.Defective advenovirus vector system is the only one which was approved to use in clinic. It can not only efficiently infect target cells, but also effectively express interest genes while virus itself don't integrate into chromosomes, so the biology safety is perfect. Because of its advantages as above, it has great development potentiality and application prospect.5. Antisense hTERT RNA could efficiently inhibit tumor growth in vivoRecombinant adenovirus could significantly inhibit hepatoma cells growth in vivo. The effect depends on the time and drug administrations: 1010 MOI /mouse, once a week four 4 times, tumor cells growth could be inhibited. Null mice 72h postinjection with 2.2.15 cells were injected with drugs only once, the tumor-inhibited effect was better than given repeatly. The results demonstrated that recombinant adenovirus could effectively therapy early-period tumors and remainder tumor cells after operation.6. Effect of two types of antisense-RNAApoptosis of HepG2-asmyc cells infected with pAd-ashmyc and HepG2-asmyc cells control was 18.9% and 13.1% respectively. The telomerase activity was 2.058 and 2.39 in different two types of cells respectively.