| Liver cancer is one of the most common malignancies, and the primary carcinoma of the liver is the fifth most common malignancy in the world and is the third cause of cancer-related mortality in our country. The patient’s five year viability is less than 5% although researchers spend decades on it. Gene therapy has been a new way for malignancy treatment since 1980s. It is a new biomedical technology which induce normal human gene someway into human target cells to correct the gene flaw so that to attain a goal of treatment. However, liver cancer is a disease caused by multiple gene disorder such as activation of oncogene, silence of tumor suppressor gene, over-expression of anti-apoptosis gene, chaos of cell cycle and increase of angiogenesis factor instead of a single gene. As a result, the traditionally gene therapy cannot fulfill the complexity and diversity of the liver cancer.microRNA (miRNA), a kind of small RNA discovered in the new century, can regulate the gene expression in translation lever. Recently,researchers found that microRNA is closely related to the malignancy genesis, growth and metastasis. miR-34a has been proved to be a key factor to play the role of tumor suppression for an important suppressor oncogene p53. Moreover, miR-34a can also make a positive feedback cycle to activate p53 and they together play a role in tumor suppression. Inducing miR-34a is effective to suppress and kill tumor cells but this way lack of durability and continuity. This study constructs a dual-gene target oncolytic adenovirus vector AdCN205-miR-34a to take the gene of miR-34a. This virus use hTERT promoter and knockdown of 24bp in virus E1A-CR2 sequence to target tumor cells and use RNA polymerase (pol) II promoter to control the expression of miR-34a, which can express mature miR-34a especially in tumor cells. On the other hand we also construct an adenoviral vector AdCN205-IL-24-miR-34a which can coexpress miR-34a and a traditional oncogene IL-24. With such adenovirus, we can successfully raise the expression level of miR-34a and IL-24 in tumor cells. The expression of miR-34a can reduce the expression of anti-apoptosis factor Bcl-2, which can make IL-24 more efficient to kill tumor cells. Meanwhile, the high expression of miR-34a can also suppress the expression of silent information regulator 1 (SIRT1), which can suppress the angiogenesis of the tumors. The cytotoxicity experiment result suggested that AdCN205-IL-24-miR-34a can efficiently kill liver cancer cell lines but have little effect to normal cell lines. The result of the xenograft tumor experiment in nude mice showed that such virus can suppress the growth of xenograft tumor and no mice have been killed during the experiment. The construction of such viral system has exploited a new way for liver cancer gene therapy.On the other hand, we have also constructed a non-replicate adenovirus Ad-miR-34a which used RNA polymerase (pol) II promoter to control the expression of microRNA specifically in tumor cells. Such adenovirus has mild effect to cells because it cannot replicate in cells. As a result, the effect to cells is mainly caused by genes carried by the vector. This vector can be used in detecting the effect of microRNA to cell-signal passway by examining the change of protein expression after infected with virus so that to confirm the target of microRNA in vivo, which has provided an effective implement for microRNA research. |
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