127 MicroRNA-218: A Key Regulatory Factor for RUNX2

Thursday, March 22, 2012: 8 a.m. - 9:30 a.m.
Presentation Type: Oral Session
A.G. SPEER1, A.C. CAVENDER1, B. AMENDT2, and I. GAY1, 1Periodontics, University of Texas - Houston/Health Science Center, Houston, TX, 2Center for Environmental & Genetic Medicine, Texas A&M Health Science Center, Houston, TX
Cell-fate determination are mediated by master regulatory transcription factors such as NANOG, OCT4 and RUNX2, which integrate multiple cell-signaling inputs in a temporal basis and generate epigenetic shifts to modulate gene expression. Advances in tissue engineering strategies include introduction, silencing, and/or manipulation of cell signals to predictably induce phenotypic changes. RUNX2 is an essential protein for osteoblastic differentiation and acts as a regulatory factors involved in skeletal gene expression; however, there is no data on the role of microRNAs (miRNA) on RUNX2 activity during dental stem cells (DSC) differentiation.

Objectives: Elucidate the role of miRNA-218 on RUNX2 in DSC under osteoinductive conditions.

Methods: Tissue samples of periodontal ligament, dental pulp, and attached gingiva were harvested from healthy discarded 3rd molars. Cells were FACS sorted to obtain a STRO-1+ enriched cell population. Total RNA was extracted at day 0 (D0) to profile miRNA expression and qRT-PCR was performed to demonstrate OCT4A/NANOG activity. DSC were cultured for 0, 7, 14, 21 and 28 days under osteogenic conditions or control. qRT-PCR was performed at each time-point to evaluate RUNX2 activity. When RUNX2 reached the highest expression, total RNA was collected for miRNA analysis. Alizarin red staining was performed at D28 to confirm mineralization. qRT-PCR and Western Blot tests were used to confirm the correlation between hsa-mir-218 and RUNX2. Human bone marrow stem cells were used as positive control for all the experiments.

Results: qRT-PCR results from OCT4A/NANOG confirmed undifferentiated status of DSC at baseline. qRT-PCR results for RUNX2 demonstrated osteogenic induction, which was confirmed using alizarin red.  Microarray of 765 miRNA indicated a 3-6 decrease in hsa-mir-218 in all DSC upon differentiation. qRT-PCR and Western Blot demonstrated an increase in RUNX2 associated to hsa-mir-218 downregulation.  

Conclusion: In the process of DSC differentiation, MiRNA-218 upregulates RUNX2 by targeting the 3′-untranslated region of the mRNA.


Keywords: Cell biology, Gene expression, MicroRNA, Mineralization and Regeneration