Abstract
Precartilaginous stem cells (PCSCs) are adult stem cells that can initiate chondrocytes and bone development. In the present study, we explored whether miR-132/212 was involved in the proliferation of PCSCs via Hedgehog signaling pathway. PCSCs were isolated and purified with the fibroblast growth factor receptor-3 (FGFR-3) antibody. Cell viability, DNA synthesis and apoptosis were measured using MTT, BrdU and flow cytometric analysis. The mRNA and protein expression were detected by real-time PCR and Western blot, respectively. The target gene for miR-132/212 was validated by luciferase reporter assay. Results showed that transfection with miR-132/212 mimic significantly increased cell viability and DNA synthesis, and inhibited apoptosis of PCSCs. By contrast, miR-132/212 inhibitor could suppress growth and promote apoptosis of PCSCs. Luciferase reporter assays indicated that transfection of miR-132/212 led to a marked reduction of luciferase activity, but had no effect on PTCH1 3′-UTR mutated fragment, suggesting that Patched1 (PTCH1) is a target of miR-132/212. Furthermore, treatment with miR-132/212 mimics obviously increased the protein expression of Indian hedgehog (Ihh) and parathyroid hormone related protein (PTHrP), which was decreased after treatment with Hedgehog signaling inhibitor, cyclopamine. We also found that inhibition of Ihh/PTHrP signaling by cyclopamine significantly suppressed growth and DNA synthesis, and induced apoptosis in PCSCs. These findings demonstrate that miR-132/212 promotes growth and inhibits apoptosis in PCSCs by regulating PTCH1-mediated Ihh/PTHrP pathway, suggesting that miR-132/212 cluster might serve as a novel target for bone diseases.
Highlights
Precartilaginous stem cells (PCSCs), a type of adult stem cells, are discovered in the epiphyseal organ in embryonic limbs
We for the first time demonstrated that miR-132/212 cluster contributes to the proliferation and inhibits apoptosis of PCSCs by targeting Indian hedgehog (Ihh)/parathyroid hormone related protein (PTHrP) signaling pathway
Our evidence suggests that Ihh/PTHrP signaling cascade is important for miR-132/212-induced growth in PCSCs
Summary
Precartilaginous stem cells (PCSCs), a type of adult stem cells, are discovered in the epiphyseal organ in embryonic limbs. PCSCs play critical roles in endochondral ossification, limb growth, cartilage development and repair of damaged articular cartilage [1,2]. MicroRNAs (miRNAs) are a group of small, noncoding RNAs that play critical roles in diverse biological processes, such as cell growth, differentiation, apoptosis and angiogenesis [3]. It is well known that miRNAs lead to post-transcriptional silencing of their target mRNAs through complementary binding to the 3 untranslated region (UTR) of mRNA targets. Previous studies have suggested that miRNAs are required for skeletal development using mice with conditional knockdown of Dicer in chondrocytes of the growth plate [4,5]. Accumulating evidence indicates that miRNAs play an important role in regulating multiple developmental processes in diverse
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