| 研究実績の概要 |
The critical implication of microRNAs (miRNAs) in cancer progression has been well described. Notably, epigenetic silencing of tumor-suppressor miRNAs through DNA hypermethylation is considered a mark of human malignancies. In this project, our goal was to develop a reliable system for targeted demethylation and reactivation of specific tumor-suppressor miRNAs by epigenome editing using a dCas9-Tet1 system.
(i) We already identified a critical miRNA, which is epigenetically controlled and silenced in liver cancer cells, and designed small base-pairing guide RNAs (gRNAs) that specifically target the DNA elements with methylations marks within the promoter region of this miRNA.
(ii) By using the dCas9-Tet1 system, we successfully induced the specific demethylation (epigenetic unmasking) and re-expression of the target tumor-suppressor miRNA. To achieve high demethylation rates and expression levels, we performed single-cell cloning after dCas9-Tet1 delivery. In this way, we obtained several cell lines with expression levels of the miRNA similar to those observed in normal hepatocytes, which is critical for functional analyses.
(iii) Potential targets of the reactivated miRNA have been characterized using microarray analysis. In particular, two targets (GLUT3 and PCLAF) have been validated in hepatic cell lines using qPCR and 3’-UTR assay.
(iv) Functional analysis after miRNA epigenome editing is ongoing. In particular, we are analyzing cell growth and drug response in hepatic cell lines.
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| 今後の研究の推進方策 |
First, functional analysis after miRNA epigenome editing will be continued to evaluate the tumor phenotype status of miRNA epigenome-edited cells. We are planning to extend the miRNA editing procedure to other miRNAs and other cancer cell types. We have already identified a series of candidate miRNAs in pancreatic cancer cells. Next, epigenome editing will be challenged in vivo using animal tumor growth models. Last, our results will be presented at international conferences next year and data summarized for publication.
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| 次年度使用額が生じた理由 |
The project will require consumables and materials for cell culture, molecular biology (cloning, DNA work, miRNA and mRNA work, western blotting, etc), and DNA methylation analysis, such as bisulfite treatment, COBRA, and sequencing (consumables expenses, excluding animal experimentation (\900,000 and \500,000 for FY2021 and 2021, respectively). A part of the budget will be allocated to animal experimentations (consumables expenses; \300,000, and \250,000 for FY2021 and 2022, respectively).
The budget will also cover the costs of working meetings with Dr. Hatada at Gunma University (domestic travel expenses; \25,000 per year) and scientific communications during international conferences to disseminate the research findings (overseas travel expenses for FY2022). Last, publication fees in international peer-reviewed journals will be covered by the applied budget (miscellaneous expenses for FY2022).
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