Abstract 2041: PDGF inducible microRNAs regulate gene expression in cancer cells

Abstract

Abstract Background: Platelet derived growth factor (PDGF)-regulated signaling plays a critical role in oncogenesis. By binding to the PDGF receptors, the Akt, Src and MAPK pathways are activated to regulate genes expression and cellular physiological processes. There are subtle functional differences between the effects of specific PDGF ligands (e.g. AA, BB), which are incompletely understood. MicroRNA(miR)s are single-stranded, 21-23 nucleotide regulatory RNAs that repress target gene expression either by inhibiting protein translation or by promoting target mRNA degradation. The hypothesis tested in this study was that PDGFs regulate genes expression by modulating microRNAs level in cancer cells. Methods: MicroRNA microarrays were performed to identify miR alterations after treatment of U118 glioblastoma cells with PDGF-AA and BB. Several time points were tested: 2, 6, 12, and 24 hour. Significance Analysis of Microarrays (SAM) was performed to compare expression level between different time points. Q-PCR was conducted to confirm the microarrays data. Pre-miR probe/miRCury LNA probe were transfected into cancer cells to over-express/knock down selected miRs. Western blots were used to detect miRs’ target proteins. Results: Candidate PDGF-inducible/repressible miRs were identified based on significant change in expression level after AA/BB treatment (p-value <0.01). Expression levels of 7 miRs were altered by PDGF AA and expression levels of 14 miRs were altered after PDGF BB treatment. By Q-PCR we showed that let 7d was repressed by PDGF-AA, but not by PDGF-BB in cancer cells (U118, C272 and U87) and in fibroblasts (WI38). Cyclin D1 is a known target of let7d. By overexpression and knock-down strategies we show that PDGF-AA induced cyclin D1 expression is mediated via repression of let7d. Q-PCR confirmed that miR-146b and miR-106b are inducible by PDGF-BB, but not by PDGF-AA in cancer cells (U118, C272 and U87) and in fibroblasts (WI38). MiR-146b induction by PDGF-BB is modulated via MAPK-dependent induction of Fos. A Fos binding element was identified in the promoter region of miR146b and Fos binding to this region in the presence of PDGF-BB was confirmed by chromatin immunoprecipitation. Several targets of miR146b are altered by PDGF in cancer cells. Conclusions: Our data demonstrate for the first time that PDGF signaling regulates expression of several critical oncogenic miRs which in turn alter target gene expression and function. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2041.