Study On Gene Therapy Of Sirna Targeted At Telomerase Of Hepatocellular Carcinoma Transduced By Lentivirus In Vitro

Hepatocellular carcinoma (HCC) is the most common malignant disease in Asia. Most HCC have developed to the advanced stage when patients get diagnosed at the first time. The conventional treatments of HCC, including surgery, radiation therapy and chemotherapy, have not yet getten the satisfied efficacy in patients. Therefore, clear-cut its etiopathogenesis and developing effective therapeutic approach would be a urgent project in treatment of HCC.With the development and application of molecular biology in tumorrigenesis, it has been demonstrated that the development of malignant tumors is due to the activation of protooncogenes and inactivation of tumor suppressor genes, which lead a out of control on cell proliferation and apoptosis, and a deficit of normal life span of cells resulting in cellular immortality and tumorigenesis. Recently, the studies showed that cellular immortality was caused by the activated telomerase. The role of telomerase is to maintain telomeric length and promotes cell proliferation potential. The activated telomerase is believed to be a critical event in cellular immortility. Telomere is the component of chromosome ends in eukaryotic cells. Just as the hat it consists of species specific tandem repeats of simple sequences and binding proteins Human and other mammals contain 5'-TTAGGG-3' repeats. In 1984, Greider and Blackburn found the activity of telomerase in Blackburn laboratory. In 1989, Morin found the telomerase in human breast cancer cell"Hela". Telomere length is progressively shortened in normal cells due to "end replicative problem" untill senescence, while it is short and stable in tumor cells. Telomerase is a special reverse transcriptase which contains both essential proteins and RNA component. The RNA component is used as the template for synthesizing the telomeric repeats to lengthen telomere. Telomerase expression has been detected in more then 90% of tumors, but it is absent in most normal somatic tissues. The higher the degree of malignancy of tumors is, the higher the telomerase activity is.Human telomerase contains three major subunits, RNA (hTR), human telomerase catalytic subunits (hTERT), and telomerase-associated protein (TP1), have been identified recently. hTERT was cloned in 1997, its template contains 11 nucleotides. In 1999, the hTERT gene promoter was cloned, its template contains 1.7bp nucleotides, with its core is -211~+40. Recently researches found that hTERT mRNA had no expressed in most normal tissue (heart, brain, breast, liver, skeletal muscle, prostate and placenta) except thymus, testis and small intestine et al. But some researches also found that hTERT mRNA was expressed in nearly all Hepatocellular carcinoma (HCC) as well as other cancers. And the level of hTERT mRNA was higher in malignant tumors than in normal liver tissues, and correlated positively with the degree of malignancy of tumors. hTERT is similar with elementary eukaryotic telomerase reverse transcriptase in sequence similarity. The hTERT is the catalytic subunit of telomerase. Its expression is usually parallel with telomerase activity. The hTERT is the telomerase proteinum catalytic subunit, which is expressed in proliferative cells, also is the rate-limiting step of telomerase activation and cell cancerization. To restrain the expression of hTERT could effectively shutdown the growth of HCC cells and deduce its apoptosis. So the hTERT has been the ideal target of the gene therapy to the HCC cell.Currently treatment prescriptions in gene therapy for HCC include immunopotentiation therapy; gene mediated pre-enzyme drug therapy; gene replacement and anti-sense gene therapy. These methods mainly use exogenous gene which was imported into target cells to rectification cell gene deviancy in order to achieve goals of treatment.Some researches found that a few dsRNA (double-stranded RNA) can specifically inhibit the expression of target gene efficiently and degraded mRNA. It is named as RNAi (RNA interference), belonging to PTGS (post-transcriptional gene silencing). This phenomenon generally emerges in eukaryotic cells and vitro cells, for successful target validation in therapeutic approaches in gene silencing.The key of gene therapy is to have gene highly express in target cell, which is mediated by ideal carrier efficiently. So there is a high requirement for gene carrier. The current carriers include non viral vector and viral vector. However, the former one has a high safety advantage, with a low efficiently transfect rate. Leniviral Vector (pLenti-GFP) is a high rate of transduction virus vector with low cytotoxicity. The vectors can not only transduce proliferating cells, but also transduce quiescent cell, such as neuron cells, without any cytotoxicity.In this study, we constructed the gene vector pLenti-GFP containing the hTERT gene and transduced it into HepG-2 cell in vitro. We have studied the inhibitory effects of hTERT-siRNA to HepG-2 cells (one kind of human HCC cells) by using RNAi to inhabit the expression of hTERT. Because of the unfavorable outcome of conventional therapy for malignant HCC, it is top priority to develop new therapeutic strategy for HCC. Gene therapy has been studied for the treatment of HCC. The application the hTERT-siRNA of gene therapy has been limited due to selection of suitable gene transfer vector and transfective efficiency of it. So, approach some high efficiency and hypotoxic vector to mediate gene therapy is another hot spot.Since the effectiveness of cancer gene therapy is mainly determined by selective vector, the majority of the research in this area has been focused on designing an efficient and safe vector system. At present, viral vector, especially pLenti-GFP (Leniviral vectors) appears to be a promising solution for cancer gene therapy.In our study, the hTERT-siRNA was used as therapeutic gene, which is linked to the GFP reporter gene in plenti-GFP vector, then was transduced into HepG-2 cells. No-load group and blank group are determined as control. The transduce rate was detected by fluorescent microscope. The results showed that the rate of transduction was more than 90% in plenti-GFP gene transduced group. In addition, the growth of cells in No-load group and blank group is similar, which indicated that the plenti-GFP is a vector with lower toxicity. It was demonstrated that pLenti-GFP can be used as a promising vector in gene therapy. The hTERT (human telomerase reverse transcriptase) is the telomerase proteinum catalytic subunit, which is expressed in proliferative cells, also is the rate-limiting step of telomerase activation and cell cancerization. To restrain the expression of hTERT could effectively shutdown the growth of HCC cells and induce its apoptosis. So the hTERT has been the ideal target of the gene therapy to the HCC cell. RNAi (RNA interference), for successful target validation in therapeutic approaches in cancer and many other diseases, it has emerged as a useful tool to specifically inhibit the expression of a selected factor and study its function in the disease process. Gene silencing by RNA interference requires the application of double-stranded short inhibitory RNA (siRNA) that is designed to bind a selected mRNA sequence after intracellular complexation with the RNA-induced silencing complex (RISC). Upon binding of the specific sequence the RISC complex cleaves the targeted mRNA which is subsequently degraded and ultimately results in highly efficient and selective protein synthesis inhibition. Only very few siRNA copies per cell are required to mediate efficient gene silencing which has raised this technique as a widely approached attractive therapeutic strategy for various diseases including cancer. Use 21nt siRNA can silence the expression of hTERT efficiently.In part I, we have constructed the vector pLenti-GFP containing with hTERT, which had a high efficient transduction. Telomerase activity and hTERT were highly expressed in HCC after transduced by this vector. It suggests that hTERT maybe used as a target for RNAi gene therapy. In this part, hTERT-siRNA was transduced into human HepG-2 HCC cells. The telomerase activity and the expression of gene were examined using the same method mentioned above; the cell proliferation and apoptosis were detected by MTT assay and RT-PCR assay. The results were as follows: Proliferation activity of HepG-2 cells transduced with hTERT-siRNA was inhibited significantly and the inhibition rate was time dependent. The inhibitory rate was 57.5% at 7 day after transduction with hTERT-siRNA. Compared with control and no-load group, the telomerase activity and the expression of hTERT mRNA was lowered significantly in HepG2 cells transduced with hTERT-siRNA. There was no change in no-load group and control groups.These results indicated that with hTERT-siRNA transduction, expression of hTERT mRNA was decreased, telomerase activity was inhibited, and proliferation of tumor cells was suppressed, moreover apoptosis of tumor cells was induced.PARTⅢ: study on inhibitory effects of hTERT-siRNA to HepG2 cells mediated by pLenti-GFP in vivoWe have constructed the vector pLenti-GFP with a high efficient transduction. In this part, we have performed the study on inhibitory effects of hTERT-siRNA to HepG-2 cells mediated by pLenti-GFP in vivo. HepG-2 cells transduced with hTERT-siRNA and without hTERT-siRNA were implanted into subcutaneous of nude mice respectively. During the course of the study, primary tumor sizes were estimated by each other 3 days with measuring the perpendicular minor dimension (W) and major dimension (L) using sliding calipers. Approximate tumor volume was calculated by the formula (W2x L) x 1/2. The study was ended at day 30; all animals in each group were sacrificed. The primary tumors were excised, collected and fixed in paraformaldehyde and embedded in paraffin blocks for histology analysis. The apoptosis of cells were detected by TUNEL assay. As the results: Proliferation activity of HepG2 cells transduced with hTERT-siRNA was inhibited significantly and the inhibition rate was time dependent. HE staining indicated that hTERT siRNA induced cell necrosis, and inflammatory cell infiltrate to the tumor cells was displayed. TUNEL assay also demonstrated that hTERT siRNA induced cell apoptosis.In summary, our study demonstrated that the hTERT is a promising molecular target for HCC gene therapy. The combination of the efficient transduction of pLenti-GFP and the effective targeting of siRNA for hTERT will provide a potential therapeutic approach to treating HCC and also extend the application of siRNA to nano-based therapies and to basic research. Meanwhile, there are many questions need to be deeply understood such as regulation of hTERT gene expression and induction of apoptosis in tumor cells.