MICRORNA-302A-3P REGULATES PROSTAGLANDIN E2-INDUCED RANK LIGAND EXPRESSION IN HUMAN MANDIBULAR BONE-DERIVED CELLS

Abstract

Receptor activator of nuclear factor kappa-B (RANKL) plays an essential role in osteoclastogenesis. MicroRNAs (miRNAs) regulate gene expression at the post-transcriptional level in several biological processes including osteoclastogenesis. This study aimed to search for the candidate miRNA that regulates RANKL expression in human mandibular bone-derived cells (HMBCs) by using the inflammatory miRNAs PCR array (Qiagen). To mimic inflammation, PGE2 treatment increases RANKL mRNA and protein in HMBCs whereas interferon-γ (IFNγ) suppresses RANKL expression. The miRNA profile of HMBCs in these conditions shows that miRNA-302a-3p, is down-regulated when RANKL increased, and up-regulated with RANKL suppression, and this result is confirmed by using qPCR. By using TargetScanHuman 7.0, the target of miRNA-302a-3p is predicted to be PRKACB mRNA that encodes catalytic subunit of PKA signaling. As RANKL expression in HMBCs is regulated through PKA signaling, miRNA-302a-3p therefore may play a role in this mechanism. To investigate its mechanism in RANKL expression, PGE2-treated cells, that contain diminished level of miRNA-302a-3p, are transfected with miRNA-302a-3p mimic. When miRNA-302a-3p level is restored, HMBCs demonstrate decreased level of RANKL mRNA and protein in the presence of PGE2. By contrast, IFNγ-treated cells show low level of RANKL with up-regulation of miRNA-302a-3p. Therefore, the transfection of miRNA-302a-3p inhibitor can suppress miRNA-302a-3p expression and increase RANKL mRNA and protein in HMBCs. Our results indicate that the level of miRNA-302-3p affects RANKL mRNA and protein expression in HMBCs. Since the target of miRNA-302a-3p may be PRKACB mRNA, when available, miRNA-302a-3p may decrease RANKL expression in HMBCs through suppression of cAMP/PKA signaling. The RANKL release by HMBCs may therefore influence the osteoclast differentiation and alveolar bone resorption during inflammation.