Abstract
Bone morphogenetic protein 2 (BMP2) induces effective chondrogenesis of mesenchymal stem cells (MSCs) by promoting Sox9 expression. However, BMP2 also induces chondrocyte hypertrophy and endochondral ossification by upregulating Smad7 expression, which leads to the disruption of chondrogenesis. In addition, Smad7 can be inhibited by Sox9. Therefore, the underlying mechanism is not clear. Currently, an increasing number of studies have shown that microRNAs play a pivotal role in chondrogenic and pathophysiological processes of cartilage. The purpose of this study was to determine which microRNA is increased by Sox9 and targets Smad7, thus assisting BMP2 in maintaining stable chondrogenesis. We found that miR-322-5p meets the requirement through next-generation sequencing (NGS) and bioinformatic analysis. The targeting relationship between miR-322-5p and Smad7 was confirmed by dual-luciferase reporter assays, qPCR, and western blotting (WB). The in vitro study indicated that overexpression of miR-322-5p significantly inhibited Smad7 expression, thus causing increased chondrogenic differentiation and decreased hypertrophic differentiation, while silencing of miR-322-5p led to the opposite results. Flow cytometry (FCM) analysis indicated that overexpression of miR-322-5p significantly decreased the rate of early apoptosis in BMP2-stimulated MSCs, while silencing of miR-322-5p increased the rate. A mouse limb explant assay revealed that the expression of miR-322-5p was negatively correlated with the length of the BMP2-stimulated hypertrophic zone of the growth plate. An in vivo study also confirmed that miR-322-5p assisted BMP2 in chondrogenic differentiation. Taken together, our results suggested that Sox9-increased miR-322-5p expression can promote BMP2-induced chondrogenesis by targeting Smad7, which can be exploited for effective tissue engineering of cartilage.
Highlights
Traumatic or degenerative cartilage defects are a challenging clinical issue, as cartilage tissue is devoid of vascular, neural, or lymphatic structures [1]
AdBMP2 and AdSox9 were flagged with green fluorescent protein (GFP), and AdshSox9 was labeled with red fluorescent protein (RFP)
Gene expression detected by qPCR at days 3, 6, 8, and 10 after infection revealed that silencing Sox9 inhibited Bone morphogenetic protein 2 (BMP2)-induced Sox9 and COL2A1 expression while promoting BMP2-induced Smad7 expression (Figures 1(a)–1(c))
Summary
Traumatic or degenerative cartilage defects are a challenging clinical issue, as cartilage tissue is devoid of vascular, neural, or lymphatic structures [1]. The above characteristics contribute to the poor self-healing capacity of cartilage. Cartilage needs to be reestablished once injured [2]. Mesenchymal stem cells (MSCs) have been identified as ideal seed cells in cartilage tissue engineering due to their chondrogenic differentiation potential [2,3,4]. Bone morphogenetic protein 2 (BMP2), a member of the transforming growth factor beta (TGF-β) superfamily, is a potent growth factor for the induction of MSC chondrogenic differentiation [5,6,7]. BMP2 alone cannot achieve stable chondrogenesis, as it stimulates chondrogenic hypertrophic differentiation and endochondral ossification, which
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