| Project/Area Number |
17591740
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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 |
Obstetrics and gynecology
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| Research Institution | Okayama University |
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Principal Investigator |
YOSHINOUCHI Mitsuo Okayama University, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Lecturer (50261235)
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| Project Period (FY) |
2005 – 2006
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| Project Status |
Completed (Fiscal Year 2006)
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| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2006: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2005: ¥2,500,000 (Direct Cost: ¥2,500,000)
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| Keywords | HPV / E6 / E7 / RNAi / siRNA / cervix cancer / cell proliferation / HPV 16 / HPV 18 |
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| Research Abstract |
Small-interfering RNAs (siRNAs) have been recently shown to be the most powerful tools that can silence gene expression by degrading mRNA in a sequence-specific manner. K. Tahira, et. al. firstly demonstrated gene silencing mediated by DNA methylation can be induced by dsRNAs in mammalian cells (Nature. 431:211-7. 2004.). Synthetic siRNAs targeted to CpG islands of the E-cadherin or erbB2 promoter induced significant DNA methylation and histone H3 lysine 9 methylation in MCF-7 cells, and expression of these genes were repressed at the transcriptional level. We have reported that down-regulation of HPV E6 and E7 expression by siRNAs led to retarded growth of HPV-positive cervical cancer cells, and proposed that siRNA-induced E6 and E7 silencing has a major therapeutic potential. E6 and E7 expression was regulated by the enhancer and p105 promoter in LCR of HPV. The object of this project is to determine whether DNA methylation can be induced by siRNAs targeting CpG islands of LCR. Ten siRNAs covering CpG sites were synthesized and introduced into HPV16-positive SiHa cells. No growth inhibition was observed at all.
During these efforts, the controversy over the work of Tahira was reported. On Mar. 30, 2006, an investigation panel from Tokyo University made an announcement that most of the results of Tahira's RNA paper including the Nature paper regarding DNA methylation had been intentionally fabricated. It was then concluded that DNA methylation cannot be induced by siRNAs targeting CpG islands of LCR. The BamHI fragment of the HPV LCR was ligated at the BamHI site of the pEGFP-1 plasmid, and the resultant plasmids were transfected into HPV-negative C33a cells. All these preparations to reveal DNA methylation and histone I-13 lysine 9 methylation were wasted.
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