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Study on Gene Therapy of SiRNA Targeted at Telomerase of Hepatocellular Carcinoma Transduced by Lentivirus in Vitro

Author: ZhangYong
Tutor: SunBeiCheng
School: Nanjing Medical University
Course: Liver surgery
Keywords: Hepatic carcinoma Telomerase Human telomerase catalytic subunit Gene Expression RNA interference Small interfering RNA Lentiviral vectors RT-PCR TUNEL method. Human hepatoma cell line HepG2
CLC: R735.7
Type: Master's thesis
Year: 2009
Downloads: 65
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Abstract

Liver tumor is the most common malignancy in Asia, initial diagnosis most advanced. Traditional surgery, radiotherapy and chemotherapy treatment ineffective. Therefore explicitly to its development mechanism occurs explore the effective treatment has become an urgent problem of today's general surgery. With the development of molecular biology, it is gradually recognized that the mechanism of tumorigenesis is mainly out of control due to the activation of oncogenes and inactivation of tumor suppressor genes and lead to tumor cell proliferation, apoptosis defects as well as normal cells limited life cycle defects cell immortalization, followed by the formation of tumors. At home and abroad study found that the the cells immortalized main telomerase within cells activated in the role of certain factors, thereby increasing telomere sequence by telomerase stable telomere length, and thus the ability to increase cell proliferation and their immortalization. Thus, activation of telomerase can be said is a key step in cell immortalization. Telomeres are the ends of a region of linear chromosomes of eukaryotic cells, composed of many kind of the case of a specific simple repetition of the DNA sequence and its specific binding proteins, it is to provide a protective \In humans and other vertebrates repeat sequence 5'-TTAGGG-3 '. Blackburn's laboratory in Berkeley, Greider and Blackburn in 1984 for the first time from Tetrahymena cells extract telomerase activity. 1989 Morin was first found in human cancer cell lines Hela cells telomerase. Telomerase is a ribonucleoprotein (ribonucleoprotein RNP), is a special kind of anti-transcribed RNA-dependent DNA polymerase, RNA and protein composite body, in order to own RNA as template, and by reversing the recorded while the synthesis of telomeres, the telomeric repeat fragment was added to the ends of chromosomes. Therefore, it solves DNA semiconservative end replication problem in copy mode. Counter 1994 First detected in metastatic ovarian cancer among the telomerase activity. Reported a lot of literature on the relationship between the tumor and the telomerase scholars from various countries in recent years, found that normal body cells, in addition to higher end update organization of embryonic cells and germ cells and in most developing proliferative capacity tablets of enzyme activity, most telomerase-negative, telomerase activity detection by more than 5000 cases of tumor specimens found, of which approximately 85% were positive telomerase-positive, more than 90% of the malignant tumors, and end granzyme activity and increased with increasing degree of malignancy of. Human telomerase including RNA (human of telomerase RNA, hTR) and the catalytic subunit (human telomerase reverse transcriptase, hTERT) and related protein 1 (of telomerase associated protein 1, TP1). hTERT is a reverse transcriptase (reverse transcriptase, RT) family members, about 40kb size single-copy gene, located in the 5p15.33. Catalytic subunit gene first Lendvay found in yeast. Encoding hTERT gene was cloned in 1997. hTERT promoter sequence was cloned in 1999, there is 1.7kb long, wherein 251bp (-211 ~ 40) is the core part. Recent studies have found that hTERT mRNA expressed at low levels in several normal tissues in the thymus, testis, small intestine, in most normal tissues (heart, brain, breast, liver, skeletal muscle, prostate, placenta, ovary) are not detected. However, the detection of a variety of tumor tissue is found in most of the tumor tissues, hTERT mRNA expression of hTERT mRNA can be detected in telomerase-positive tumor. HTERT mRNA in liver cancer: of hTERT mRNA in hepatoma cells in the presence of high expression of hTERT mRNA and hTERT protein expression levels are closely related to the degree of malignancy of the liver cancer, and increased with the increase in the degree of malignancy of the tumor and normal The liver tissue is not the expression of hTERT. Currently considered hTERT is the catalytic protein component of telomerase, telomerase activity and the rate-limiting step of cancerous cells, and inhibition of its expression to be effective in inhibiting the growth of tumor cells and the induction of apoptosis, can be used as liver cancer gene therapy ideal target. The current program of gene therapy for liver cancer including immune enhancement therapy, gene-mediated enzyme prodrug therapy, tumor suppressor gene replacement therapy and antisense therapy. These methods are mainly through the expression of exogenous gene is introduced into host target cells, so to correct the cell genetic defects, the purpose of treatment of disease. With the development of molecular biology techniques, gene therapy technology has been optimized and improved. Recent studies found that some of the small double-stranded RNA (double-stranded RNA, dsRNA) can be efficiently and specifically blocking the expression of certain genes in vivo, to promote its mRNA degradation, induces cell phenotype of the specific gene deletions, such technology of RNA interference (RNA interference, RNAi), belongs to the post-transcriptional gene silencing (post-transcrptional gene silencing, PTGS). RNAi phenomenon is widely present in most eukaryotic cells, the cultured cells in the mouse and human in vitro gene expression using RNAi technique can also be successfully blocked, to achieve the cellular level of the gene knockout. The key to the success of gene therapy is the ideal carrier to efficiently target gene transfer into target cells and get a good expression, put forward higher requirements which gene therapy vectors. Commonly used carrier is divided into two major categories of non-viral vectors and viral vectors, the former has a higher security, but the transfection efficiency is poor, so in recent years people are more focused on the development and application of a viral vector. Lentivirus vector (Leniviral Vector, pLenti) is a viral vector having a higher transfection efficiency and low cytotoxicity. Can infect not only proliferating cells are also infected with non-proliferating cells and quiescent cells such as neurons, without any side effects. Based on the above, the test is based on first pLenti-GFP, to build pLenti-of hTERT-siRNA transfected human hepatoma cell line HepG2 cells in vitro inhibition of liver cancer cells with high expression of hTERT, through the mechanism of RNAi to explore on the growth of liver cancer cells inhibition. Liver cancer conventional treatments ineffective, and explore new therapeutic strategies has become imperative. Gene therapy for liver cancer has developed rapidly in recent years, and some progress. But due to the gene therapy itself some constraints, such as low carrier safety and transfection efficiency, making the development of gene therapy subject to certain restrictions. Therefore, to explore the application of some of the new hot spot for high transfection efficiency and low cell toxicity of novel targeted vectors to mediate gene product treatment for liver cancer gene therapy research. Select the appropriate gene transfer vector target genes safely and efficiently into cells, which is key to the success of gene therapy. Viral vectors in gene transfer from the current development trend presents a significant advantage, which lentiviral vectors have received increasing attention for its superior physical and chemical properties and ideal gene transfer. Studies have shown the pLenti-GFP efficiently mediated gene transfection of a variety of tumor cell lines and primary cultured cells, and has the advantage of slow release, safe and nontoxic. In this study, the use of GFP fluorescence labeled hTERT-siRNA as a therapeutic gene as a gene delivery carrier select pLenti-GFP in vitro transfection of human hepatoma cell HepG-2 by fluorescence microscopy analysis of the efficiency of transfection and empty vector control, slow pLenti-GFP cell toxicity of viral vectors for the continuous observation and analysis. This part of the study was designed to explore the feasibility of HCC gene therapy using lentiviral vectors pLenti-GFP to further the search for a better means of gene transfer for liver cancer gene therapy. : The results show that the transfection efficiency of the lentiviral vector pLenti-GFP in human hepatoma cells HepG-2 is detected by fluorescence microscopy, pLenti-GFP transfection efficiency of more than 90%, completely in vitro test that can be used for gene therapy of liver cancer. In addition, the empty vector cell growth state, apoptosis rate with blank control group without any intervention, similar, which proved pLenti-GFP is a low cytotoxicity carrier for liver cancer gene therapy research. hTERT is a telomerase catalytic subunit, often expressed in proliferating cells only, is a limiting step, telomerase activation and carcinogenesis inhibiting its expression to be effective in inhibiting the growth of tumor cells and induce apoptosis, has become ideal target for cancer gene therapy. RNAi is a sequence-specific post-transcriptional gene silencing by double-stranded RNA, can efficiently and specifically block the expression of specific genes in mammalian cells. It is imported in vivo or endogenous transcription of dsRNA generated by an RNase Ⅲ nuclease Dicer cut to 21 ~ 25nt small interfering RNA, namely siRNA, siRNA further with a variety of other protein components combine to form the RNA-induced silencing complex (RNA-induced silencing complex, RISC), and finally by the RISC mediated siRNA antisense strand with the target mRNA molecule is complementary binding and cause the cutting effect of homology of a target mRNA molecule. In the first part, we have successfully prepared with high transfection efficiency the gene therapy the carrier pLenti-GFP In this section, we design for of hTERT siRNA gene interfere with treatment of human hepatoma cell line HepG-2 cells, results, respectively, using the MTT assay and RT-PCR analysis of the rate of cell proliferation after transfection, hTERT mRNA expression, and to investigate the cell growth inhibition and its mechanism. The results showed that: of hTERT-siRNA in vitro can significantly inhibit the growth of HepG-2 cell lines, apoptosis increased inhibition increased with time, the seventh day of its cell inhibition rate of 57.5%, and the differences in the control group statistically significant. hTERT-siRNA transfected HepG-2 cells, hTERT mRNA expression was significantly reduced telomerase activity levels dropped significantly; blank group and the no-load group transfected with hTERT mRNA and telomerase activity did not change significantly. As can be seen from the above results: hTERT-siRNA can inhibit hTERT mRNA expression in tumor cells, telomerase activity levels, thereby inhibiting tumor cell growth, proliferation and promote apoptosis of tumor cells. In this section, we design for of hTERT siRNA gene interfere with treatment in vivo studies of the human hepatoma cell line HepG-2 cells. We use the HepG2 cells in nude mice were inoculated with transfected and untransfected objective gene. Long and short diameters of tumors were measured with a caliper every 3 days, and tumor volume was calculated (V = 1/2 × long diameter × short diameter × short diameter). The mice were sacrificed after 30 days, remove the tumor, fixed in paraformaldehyde embedded for histological examination. Serial sections, routine HE staining, light microscopic examination, tumor histology, TUNEL assay of apoptosis. The results show: the treatment group tumors grow slowly, with time, more and more obvious differences. Routine pathological HE staining showed necrosis increased infiltration of inflammatory cells around. TUNEL assay show an increase in apoptosis. Comprehensive experimental results of three parts, we think, hTERT is the ideal target for liver cancer gene therapy, RNAi and pLenti-GFP gene transfer technology combine the treatment of liver cancer is a practical new strategy is expected to gene therapy for liver cancer in the future. opened up a whole new technology platform has broad application prospects. At the same time, many important issues, including expression of hTERT regulatory network and its silencing induced apoptosis mechanism has yet to be a more in-depth research.

Study on Gene Therapy of SiRNA Targeted at Telomerase of Hepatocellular Carcinoma Transduced by Lentivirus in Vitro

Abstract

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