Study of the Angiogenesis Promoting Effect of Irradiation Induced Mir-17-5p in Oral Cancer Cells

Our past study has demonstrated that miR-17-5p can negatively regulate p21 expression and thus reduces the radio-sensitivity of oral cancer cells (Strahlenther Onkol. 20133; Oncotarget 2016). We also found that miR-17-5p can reduce PTEN expression and thus enhances the viability of breast cancer cells (J Cancer. 2016). These finding demonstrated that miR-17-5p plays critical role in the malignancy of human cancer cells. Interestingly, we also observed the anti tumor angiogenesis effect in the usage of miR-17-5p inhibitor. However, there is no article mentioned about the mechanism of miR-17-5p on the tumor angiogenesis. In the two-years project, we will investigate the molecular mechanism and therapeutic strategy of radiation induced miR-17-5p on the angiogenesis promoting capability of oral cancer cell line OC3(from Taiwan) and SCC4,SCC9 and SCC25 (from ATCC). The aim and study design of each year are:In the first year, we will clarify the promoting effect of miR-17-5p on angiogenesis and identify the miR-17-5p regulated specific angiogenesis protein in oral cancer cells.Major strategies and methods are:1. Collect the cell culture supernatant from irradiated oral cancer cell lines with miR-17-5p inhibitor or vehicle. The cell culture supernatant will be used in in vitro angionenesis assays to clarify the angiogenesis promoting capability.2. The cell culture supernatant that collected as described above will be used to screen the miR-17-5p regulated angiogenesis related protein by angiogenesis protein array.3. The translational regulation of miR-17-5p on specific angiogenesis related protein will be confirmed by EIA, the transcriptional regulation of miR-17-5p on specific angiogenesis related protein encoded gene expression will be confirmed by RT-PCR.4. The role of p21 or PTEN signaling in the miR-17-5p regulated specific angiogenesis related protein expression will be confirmed by p21 or PTEN siRNA strategy.In the second year, we will clarify the anti-angiogenesis effect of miR-17-5p inhibitor in the oral cancer tumor radiotherapy. Major strategies and methods are:1. Establish the tumor growth curve of OC3, SCC4, SCC9 and SCC25 cells tumor in SCID mice with control ODN or with miR-17-5p AS ODN prior radiotherapy. The tumor tissue will be collected within different time periods.2. The expression of miR-17-5p in the tumor tissue samples will be determined by QPCR, the tumor angiogenesis and cell proliferation status in the tumor tissues will be determined by IHC of neo vascular endothelial marker CD34 and cell proliferation marker Ki67, respectively.3. The quantitative results of miR-17-5p, micro-vessel density and cell proliferation index, specific angiogenesis factor expression and tumor size will be processed with statistically analysis to establish the time-course relationships that can be used to evaluate the therapeutic potential.4. The in vivo OC3, SCC4, SCC9 and SCC25 tumor model will be used to determine the therapeutic effect of radio therapy that combination with targeting miR-17-5p modulated angiogenesis factor by neutralizing antibody. 5. Confirm the clinical significance of miR-17-5p in human oral cancer by using the commercial available human oral cancer tissue cDNA arrays which have the clinical radiotherapy and prognosis data, we will analysis the miR-17-5p, and specific angiogenesis factors gene expression by QPCR.