lncRNA HHIP-AS1/HHIP modulates osteogenic differentiation of BM-MSCs by regulating Hedgehog signaling pathway

lncRNA, a type of non-coding RNA, plays an important role in the osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BM-MSCs). In this study, lncRNA and mRNA microarrays were performed to study the change of gene expression during osteogenic differentiation of BM-MSCs. We focused on Hedgehog interacting protein (HHIP), because HHIP mRNA and lncRNA HHIP-AS1 were gradually down-regulated on days 0, 7, and 14 during osteogenic differentiation. In addition, the gene coding lncRNA HHIP-AS1 is located on the anti-sense of Hhip gene, implying the potential interaction between lncRNA HHIP-AS1 and HHIP mRNA.BM-MSCs with over-expressed or silenced lncRNA HHIP-AS1 were constructed to explore the biological role of HHIP-AS1 in osteogenic differentiation. BM-MSCs were lysed to determine the alkaline phosphatase activity. Fluorescence in situ hybridization and immunofluorescence were performed to analyze HHIP-AS1, HHIP, RUNX2 and osteocalcin.Overexpression of lncRNA HHIP-AS1 increased HHIP expression, which suppressed Hedgehog signaling pathway, as indicated by the reduction of SMO, Gli1 and Gli2. The suppression of Hedgehog signal was associated with the inhibited osteogenesis. HHIP knockdown abolished the suppression of osteogenesis induced by lncRNA HHIP-AS1 overexpression. Through binding to HHIP mRNA, lncRNA HHIP-AS1 recruited ELAVL1 to HHIP mRNA, whereby increasing the mRNA stability and the protein level.This study revealed that down-regulation of HHIP due to lncRNA HHIP-AS1 reduction promoted the osteogenic differentiation of BM-MSCs though removing the suppression of Hedgehog signal.