Abstract 1796: p53 regulation of osteoblast differentiation is mediated through specific microRNAs

Abstract

Abstract We have previously shown a role for tumor suppressor gene p53 in the transcriptional activation of several bone-specific genes in osteoblast differentiation. In this study, we used a screen for microRNAs to look for ones that were p53 dependent and differentiation dependent. In a model of invitro osteoblast differentiation, our previous work has established that p53 levels generally increase around day 4 of differentiation and represent the timeline when p53 transcriptionally regulates bone-specific gene expression. In these experiments, we used MC3T3-E1 osteoblasts to stably reduced p53 levels using specific shRNAs. These cells were initially characterized for the reduction in p53 levels and then compared with MC3T3E1 cells stably transfected with a scrambled control (Control MC3T3E1 cells). MicroRNAs were isolated from day 0 and day 4 after treatment with differentiation promoting (DP) media. A microRNA profiling service utilizing a microarray detection system analyzed over 1000 different microRNAs in control and DP treated cells to determine differentiation specific microRNAs expression. This array provided us with information about a number of genes that underwent alterations during differentiation in a p53 dependent manner. We validated several of the changes using realtime PCR and selected two microRNAs to study in detail. These two microRNAs (mIR 34b and 140) were increased two fold during normal differentiation but showed a dramatic reduction in expression when p53 levels were reduced. We ectopically expressed these microRNAs in MC3T3-E1 cells and created stable lines. Cells carrying mIR34b showed a decrease in cell proliferation rates when compared to mIR140 expressing and control cells. P53 levels directly correlated with microRNA 34b expression but not mIR140. MiR 140 ectopic expression affected Bone morphogenetic protein expression (BMP2), an important bone anabolic agent that is also p53 regulated. Putative target binding sites for bone-specific transcription factors Runx2, SP7, Vitamin D receptor (VDR) were found for mIR34b and 140 and were utilized in luciferase reporter assays to confirm microRNA specific interactions. These studies provided evidence and confirmed for us that the p53 regulation of osteoblast differentiation is also mediated through specific microRNAs that directly target important bone-specific genes. Citation Format: Elisha Pendleton, Shivang Shah, Oliver Couture, Teresa Kusper, Mustafa Broachwalla, Lauren Alt, Michael Fay, Nalini Chandar. p53 regulation of osteoblast differentiation is mediated through specific microRNAs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1796.