The role and mechanism of miR-93-5p in osteogenic differentiation of rabbit bone marrow mesenchymal stem cells mediated by total flavonoids of rhizoma drynariae

doi:10.19438/j.cjoms.2023.05.004

Abstract

Abstract: PURPOSE: To investigate the role of miR-93-5p in osteogenic differentiation of rabbit bone marrow mesenchymal stem cells (rBMSCs) mediated by total flavonoids of rhizoma drynariae(TFRD). METHODS: The osteogenic differentiation of rBMSCs was induced by osteogenic medium. The osteogenic and adipogenic differentiation of rBMSCs were detected by Alizarin red S and oil red O staining, respectively. The expression of miR-93-5p was detected by fluorescence quantitative PCR (qPCR), and the expression level of FZD6, ALP, Runx2 and WNT pathway related proteins (Dishevelled and β-catenin) was detected by Western blotting. The binding of miR-93-5p and FZD6 was tested by double-luciferase reporter gene analysis. The data were analyzed with SPSS 22.0 software package. RESULTS: Compared with the control group, TFRD promoted the osteogenic differentiation of rBMSCs, inhibited the expression of miR-93-5p, and promoted the expression of FZD6 and osteogenic related genes ALP and Runx2. Double-luciferase reporter gene analysis showed that FZD6 was the downstream target gene of miR-93-5p. Overexpression of miR-93-5p inhibited the osteogenic differentiation of rBMSCs and the protein expression of FZD6, ALP and Runx2, while overexpression of FZD6 reversed the inhibitory effect of miR-93-5p. Treatment of rBMSCs with WNT pathway inhibition (KYA1797K) could reverse the promoting effect of TFRD on osteogenic differentiation, resulting in reduced osteoblast differentiation and downregulation of ALP, Runx2 and WNT pathway related proteins (Dishevelled and β-catenin). CONCLUSIONS: TFRD promote the osteogenic differentiation of rBMSCs by downregulating miR-93-5p and upregulating FZD6 to activate the WNT signaling pathway, which contributes to the osseointegration of dental implant.

Key words: Total flavonoids of rhizoma drynariae, miR-93-5p, FZD6, WNT pathway

CLC Number:

R783

SHEN Xiao-jing, LI Lin-lin, LU Yao, WANG Le-wei, YUAN Rong-tao, GUO Qing-yuan, ZHAO Peng. The role and mechanism of miR-93-5p in osteogenic differentiation of rabbit bone marrow mesenchymal stem cells mediated by total flavonoids of rhizoma drynariae[J]. China Journal of Oral and Maxillofacial Surgery, 2023, 21(5): 446-451.

References

[1] Papaspyridakos P, Chen CJ, Singh M, et al.Success criteria in implant dentistry: a systematic review[J]. J Dent Res, 2012, 91(3): 242-248.
[2] Brånemark PI, Adell R, Breine U, et al.Intra-osseous anchorage of dental prostheses. I. experimental studies[J]. Scand J Plast Reconstr Surg, 1969, 3(2): 81-100.
[3] Bosshardt DD, Chappuis V,Buser D.Osseointegration of titanium, titanium alloy and zirconia dental implants: current knowledge and open questions[J]. Periodontol 2000, 2017, 73(1): 22-40.
[4] Perez JR, Kouroupis D, Li DJ, et al. Tissue engineering and cell-based therapies for fractures and bone defects[J]. Front Bioeng Biotechnol, 2018, 6: 105.e1-105.e23.
[5] Hu Y, Mu P, Ma X, et al.Rhizoma drynariae total flavonoids combined with calcium carbonate ameliorates bone loss in experimentally induced Osteoporosis in rats via the regulation of Wnt3a/β-catenin pathway[J]. J Orthop Surg Res, 2021, 16(1): 702-710.
[6] Zhao K, Chen M, Liu T, et al.Rhizoma drynariae total flavonoids inhibit the inflammatory response and matrix degeneration via MAPK pathway in a rat degenerative cervical intervertebral disc model[J]. Biomed Pharmacother, 2021, 138: 111466.
[7] Chen GY, Chen JQ, Liu XY, et al.Total flavonoids of rhizoma drynariae restore the MMP/TIMP balance in models of osteoarthritis by inhibiting the activation of the NF-κB and PI3K/AKT pathways[J]. Evid Based Complement Alternat Med, 2021: 6634837.
[8] Mu P, Hu Y, Ma X, et al.Total flavonoids of Rhizoma Drynariae combined with calcium attenuate osteoporosis by reducing reactive oxygen species generation[J]. Exp Ther Med, 2021, 21(6): 618-628.
[9] He N, Zhang Y, Zhang Y, et al.Increasing fracture risk associates with plasma circulating microRNAs in aging people's sarcopenia[J]. Front Physiol, 2021, 12: 678610.
[10] Hui S, Yang Y, Li J, et al.Differential miRNAs profile and bioinformatics analyses in bone marrow mesenchymal stem cells from adolescent idiopathic scoliosis patients[J]. Spine J, 2019, 19(9): 1584-1596.
[11] Xue H, Tu Y, Ma T, et al.miR-93-5p attenuates IL-1β-induced chondrocyte apoptosis and cartilage degradation in osteoarthritis partially by targeting TCF4[J]. Bone, 2019, 123: 129-136.
[12] Zhang Y, Wei QS, Ding WB, et al.Increased microRNA-93-5p inhibits osteogenic differentiation by targeting bone morphogenetic protein-2[J]. PLoS One, 2017, 12(8): e0182678.
[13] Zhang Y, Zhuang Z, Wei Q, et al.Inhibition of miR-93-5p promotes osteogenic differentiation in a rabbit model of trauma-induced osteonecrosis of the femoral head[J]. FEBS Open Bio, 2021, 11(8): 2152-2165.
[14] Abuna RPF, Oliveira FS, Adolpho LF, et al.Frizzled 6 disruption suppresses osteoblast differentiation induced by nanotopography through the canonical Wnt signaling pathway[J]. J Cell Physiol, 2020, 235(11): 8293-8303.
[15] Hang K, Ye C, Xu J, et al.Apelin enhances the osteogenic differentiation of human bone marrow mesenchymal stem cells partly through Wnt/β-catenin signaling pathway[J]. Stem Cell Res Ther, 2019, 10(1): 189-198.
[16] Lu Y, Liu YK, Wan FY, et al.CircSmg5 stimulates the osteogenic differentiation of bone marrow mesenchymal stem cells by targeting the miR-194-5p/Fzd6 axis and beta-catenin signaling[J]. Environ Toxicol, 2022, 37(3): 593-602.
[17] Li S, Zhou H, Hu C, et al.Total flavonoids of rhizoma drynariae promotes differentiation of osteoblasts and growth of bone graft in induced membrane partly by activating Wnt/β-catenin signaling pathway[J]. Front Pharmacol, 2021, 12: 675470.