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.