Bone defects caused by trauma, tumor and inflammation disease seriously reduces the life quality of patients. Stem cell-based engineering has emerged as a promising and effective approach of bone regeneration (Luby et al. , 2019; Polymeriet al. , 2016). BMSCs are presently regarded as the gold standard cell source for bone tissue engineering, due to their self-renewal and multipotential differentiation capacity (Dai et al. , 2018; Fuet al. , 2019; Jing et al. , 2019). Multiple biological materials and growth factors have been implemented for osteoinduction of BMSCs (Um et al. , 2018; Zhang et al. , 2016). CORMs, a novel group of compounds that are carriers of CO (Motterlini et al. , 2002), have exhibited such potent effects as anti-inflammatory (Lee et al. , 2018), anti-apoptotic (Ulbrich et al. , 2016), neuroprotective (Ulbrich et al. , 2017), vascular function improvement (Foresti et al. , 2004; Motterlini et al. , 2002) and so on. Based on our previous study, CORM-3 promotes the osteogenic differentiation of rat BMSCs (Li et al. , 2018). However, the mechanism underlying was unknown.
miRNAs, about 22 nucleotides in length, are highly conserved endogenous RNAs. miRNAs can induce translational silence by binding to the 3′UTR of target mRNAs, thus affecting cell proliferation, differentiation, apoptosis, and ontogeny (Jackson et al. , 2007; O’Brien et al. , 2018; Wang et al. , 2019). Several studies have shown that miRNAs play an important role in the osteogenic differentiation of BMSCs (Huang et al. , 2019; Li, 2018; Li et al. , 2019; Wanget al. , 2019). Recently, researchers have uncovered CORMs mechanism associated with miRNAs (Uchiyama et al. , 2010). CORM-2 prevents TNF-α-induced endothelial nitric oxide synthase downregulation by inhibiting miR-155-5p biogenesis (Choi et al. , 2017). CORM-3 improves structural and functional cardiac recovery after myocardial injury via decreasing miR-206 expression (Segersvard et al. , 2018). However, little is known about CORMs and miR-195-5p. In the present study, we first confirmed the down-regulation of miR-195-5p in CORM-3-induced osteogenic differentiation of rat BMSCs, based on the previous gene sequencing result. Our results suggested that miR-195-5p might be involved in the CORM-3-induced osteogenic differentiation of rat BMSCs.
Runx2 is an osteogenesis specific transcription factor and plays an important role in osteogenic differentiation of BMSCs (Komori et al. , 1997; Xu et al. , 2015). As a marker for early osteogenic differentiation, it can promote the expressions of a number of downstream genes associated with osteogenic differentiation (Denget al. , 2013). OPN is a multifunctional protein mainly associated with bone formation. OPN is secreted by osteoblast, and is a characteristic phenotypic marker of osteoblast. It is stored in bone matrix, and effects the matrix mineralization (De Fusco et al. , 2017; Singh et al. , 2018). In the present study, up-regulation of miR-195-5p level suppressed the CORM-3-induced osteogenic differentiation of rat BMSCs in vitro , evidenced by the results of decreased mRNA and protein expressions of Runx2 and OPN, and matrix mineralization demonstrated. Meanwhile, down-regulation of miR-195-5p level enhanced the expression of osteogenic related factors during the CORM-3-induced osteogenic differentiation. These results suggested that miR-195-5p might be a regulator of CORM-3-induced osteogenic differentiation. However, from our experiment, the matrix mineralization was not enhanced significantly in miR-195-5p-deficient cells. This discrepancy might be due to the consideration as following. First, the mechanism underlying CORM-3-induced osteogenic differentiation is complex. Though the alizarin red staining of the mimics group was significantly decreased than that in the CORM-3 group, the scattered spots of alizarin red staining were still seen, which was significantly enhanced than that in the control group. It suggested that there might have other mechanism involved in the CORM-3-induced osteogenic differentiation of rat BMSCs. Secondly, miRNAs are complex network structure. Down-regulation of miR-195-5p via inhibitor transfectionin vitro might affect the levels of other miRNAs, which led to compensatory converse effect on osteogenic differentiation.
For further research, we first predicted the potential target gene Wnt3a of miR-195-5p by bioinformatics software. In the following experiment, up-regulation of miR-195-5p level decreased the protein expression of Wnt3a in cells. Conversely, the protein expression of Wnt3a was enhanced in miR-195-5p-deficient cells. The data of the regulatory effect of miR-195-5p on Wnt3a, together with the results from the luciferase reporter assay, demonstrated that miR-195-5p directly targeted Wnt3a.