D'Amato, Valentina (2010) Alterations of microRNAs expression profile induced by oncogenic Ras. [Tesi di dottorato] (Unpublished)


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Item Type: Tesi di dottorato
Uncontrolled Keywords: microRNA; Ras; FRTL-5
Date Deposited: 13 Dec 2010 22:30
Last Modified: 17 Jun 2014 06:02
URI: http://www.fedoa.unina.it/id/eprint/8139


Literature emphasizes the key role of miRNAs in cancer development. Moreover it has been demonstrated that oncogenes and tumorsuppressors can act also by regulating the expression of specific miRNAs. Previous results published by Landgraf et al., using the FRTL-5 ER-H-RasV12 inducible system highlighted that Ras is able to induce aberrant expression of microRNAs during the transformation of thyroid epithelial cells. Taking advantage of the inducible system FRTL-5 ER-H-RasV12 we decided to study the miRNAs involved in Ras-induced transformation. The aim of my project was to identify Ras-regulated miRNAs and study their functional or transcriptional role in transformation of thyroid epithelial cells. We decided first to validate the Landgraf’s libraries data by Real-Time PCR, later we selected microRNAs of interest to study their functional role in Ras- induced transformation. We measured the level of expression of miR15b, miR16, miR27a, miR27b, miR29b, miR99b, miR135a, miR135b, miR324-5p in inducible systems FRTL-5 ER-Ras clone (Cl11) treated with 4OHT for 24h, 48h, 72h, 96h and 7days, in FRTL-5 parental cell line and in FRTL-5 clones constitutively expressing human H-RasV12. From the screening by Real-Time PCR we identified two microRNAs particularly regulated by oncogenic Ras: miR135a resulted down-regulated in chronically transformed cells by Ras oncogene meanwhile miR21 resulted up- regulated by Ras oncogene in the inducible system. We over expressed miR135a in the chronically transformed FRTL-5 H-RasV12 clone V29 to test the possibility that miR135a rescue could interfere with at least one of the phenotypes induced by oncogenic Ras. We proceeded to analyze proliferation, differentiation and cell motility of FRTL-5 RasV12 miR135a cells. We observed a little reduction of proliferation and migration in miR135a clones compared to FRTL-5 RasV12. It is known that Ras is able to promote miR21 expression in an epithelial system we asked if this regulation was cell-type specific. The Real-Time PCR results highlights that even though the two inducible systems analyzed (FRTL- 5 ER-Ras and NIH ER-Ras) have a different basal level of miR21, in NIH ER- Ras cells 4OHT treatment do not lead to a significant increase of miR21, suggesting that Ras-induced miR21 up-regulation is cell-type specific. Once established the effects of oncogenic Ras on mature miR21 expression the following step was to study the mechanism through which this regulation takes place. Our data obtained by Real-Time PCR and chromatin immuno- precipitation (ChIP) suggests that miR21 induction by Ras in FRTL-5 cells is a transcriptional phenomenon. By luciferase assay we analyzed the miR21 promoter responsive to Ras oncogene and we verified if also its activation was cell-type specific. To this purpose we used FRTL-5, NIH3T3 and Rat2 cell lines. The data obtained suggests that even though oncogenic Ras is able to lightly regulate the activity of miR21 promoter in fibroblasts, the activation displayed in FRTL-5 is much stronger and significant. The final step was the identification of potential regulatory regions, aside from the promoter already identified, responsive to oncogenic Ras.

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