2007 Fiscal Year Final Research Report Summary
Cancer gene therapy using the let-7-inducing vector to inhibit K-ras signals
| Project/Area Number |
18591437
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
General surgery
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| Research Institution | Kagawa University |
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Principal Investigator |
ISHIKAWA Shinya Kagawa University, Faculty of Medicine, Research Associate (20363210)
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| Co-Investigator(Kenkyū-buntansha) |
HUANG Cheng-long Kagawa University, Faculty of Medicine, Associate professor (10271511)
LIU Dage Kagawa University, Faculty of Medicine, Research Associate (30314941)
YOKOMISE Hiroyasu Kagawa University, Faculty of Medicine, Professor (80231728)
UENO Masaki Kagawa University, Faculty of Medicine, Associate professor (30322267)
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| Project Period (FY) |
2006 – 2007
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| Keywords | let-7 / K-ras / gene therapy / adenoviral vector / micro RNA |
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| Research Abstract |
A reduced expression of let-7 has been reported to induce an overexpression of K-ras onco-protein. The overexpression of K-ras protein can induce tumor proliferation and angiogenesis. Therefore, we are performing an experimental study on the cancer gene therapy using let-7-inducing viral vectors. We evaluated the let-7 expression in cancer cell lines, and found that MAC10 is a cancer cell line with a reduced let-7 expression. We constructed let-7-inducing viral vectors using the shRNA (short hairpin RNA) type. We inserted the shRNA containing the let-7 sequence into pBApo-CMV vector to construct the let-7-inducing plasmid vector MIR09. The transfection of the MIR09 vector into MAC10 cells using the lipofection method demonstrated the effective induction of the let-7 : expression. Then, we constructed the let-7-inducing adenoviral vector. After the extraction of the sequence of "CMV IE promotor + shRNA" from the MIR09 vector, this was inserted into the cosmid vector pAxcwit. Then, the let-7-inducing adenoviral vector, let7-Ad5, was produced by the COS-TPC method. After confirming the structure and the transfection efficiency of this adenoviral vector, the let7-Ad5 vector was condensed and purified. Regarding the in vitro experiment of the gene therapy, the transfection of the let7-Ad5 vector into MAC10 cells induced the let-7 expression and inhibited the cell proliferation. In addition, we found the induction of the let-7 expression to down-regulate the protein expressions of K-ras and the c-Myc. We are performing additional experiments in other cancer cell lines with reduced let-7 expression. Furthermore, using the tumor-bearing nude mice made by the subcutaneous inoculation of MAC10 cells, we are studying an in vivo experiment of the let-7 gene therapy.
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