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.