The Wnt signaling pathway plays an important role in bone development, and it is strongly implicated in skeletal tissue regeneration and repair (Houschyar et al. , 2018; Xuet al. , 2014). Activating of the Wnt signaling pathway promotes the osteogenic differentiation of BMSCs in vitro (Chen et al. , 2019; Zhu et al. , 2020). Wnt signaling pathway is involved in the process of promoting fracture healing of rats with nonunionin vivo (Sun et al. , 2019). Wnts now comprise a family of secreted glycoproteins, in which Wnt3a is included (Nusse et al. , 2012; Willert et al. , 2012). The Wnt ligands bind receptors on the surface of recipient cells to activate the Wnt pathway. During the fracture repair process, the expressions of many Wnt ligands and receptors are upregulated (Xu et al. , 2014). Some miRNAs specifically interact with Wnt ligands, leading to consequent regulation on osteogenesis (Long et al. , 2017; Peng et al. , 2016). Upregulation of miR-16-2* blocks the Wnt signal pathway by directly targeting Wnt5a. miR-16-2* interferes with Wnt5a to regulate osteogenic differentiation of human BMSCs (Duan et al. , 2018). miR-196a promotes osteogenic differentiation of adipose stem cells via regulating Wnt/β-catenin pathway (Ai et al. , 2019). Therefore, we hypothesized the involvement of Wnt3a in osteogenesis. As shown in present study, the protein expression of wnt3a was enhanced during the CORM-3-induced osteogenic differentiation of rat BMSCs. Morever, overexpression of Wnt3a enhanced CORM-3-induced osteogenic differentiation, with the increased mRNA and protein expressions of Runx2 and OPN, and matrix mineralization. In contrast, the CORM-3-induced osteogenic differentiation was suppressed in the Wnt3a-deficient cells. These results suggested that Wnt3a promoted the CORM-3-induced osteogenic differentiation of rat BMSCs.
To further verify the regulation effect of miR-195-5p, rescue experiments were conducted by miR-195-5p mimics and pcDNA3.1-Wnt3a co-transfection. The data showed that the mRNA and protein expressions of Runx2 and OPN, and matrix mineralization were decreased in cells transfected with miR-195-5p mimics. However, the decreased results were rescued by miR-195-5p mimics and pcDNA3.1-Wnt3a co-transfection. The Wnt3a was presented as an obvious link of miR-195-5p to osteogenic differentiation of rat BMSCs.
CO, which is a gasotransmitter, displays many physiological roles in several organs and tissues. CO administration has therapeutic potential in many diseases (Gullotta et al. , 2012; Mitchell et al. , 2010; Segersvard et al. , 2018). CO has already been evaluated in phase I clinical testing and the feasibility and anti-inflammatory effects of low-dose CO inhalation in patients with chronic obstructive pulmonary disease have been demonstrated (Bathoorn et al. , 2007). However, the toxicity and limitation of CO gas prevented the CO application. CORMs, capable of liberating controlled quantities of CO, have been a valid alternative (Motterlini et al. , 2003). CORMs have shown a variety of pharmacological activities, with many reports on the biological applications of CORMs in inflammatory, vascular disease, organ transplantation and cancer (Adach et al. , 2019; Forestiet al. , 2008; Gullotta et al. , 2012; Motterlini et al. , 2002). CORMs, as a new type of drugs, have therefore tremendous therapeutic potential, and may become a new class of therapeutics against various diseases (Ji et al. , 2016).
In summary, the present study indicates that miR-195-5p negatively regulates CORM-3-induced osteogenic differentiation of rat BMSCs by targeting Wnt3a. Our results provide new molecule mechanism for CORMs and theoretical basis for bone tissue engineering.