Mechanism of miR-485-5p targeted regulation of WNT7B to inhibit osteogenic differentiation of bone marrow mesenchymal stem cell
10.3760/cma.j.cn121113-20230310-00105
- VernacularTitle:MiR-485-5p靶向调控 WNT7B抑制骨髓间质干细胞成骨分化的机制研究
- Author:
Zhan WANG
1
;
Aixian TIAN
;
Xinlong MA
;
Tiansheng LIU
;
Yi WANG
Author Information
1. 天津市天津医院检验科，天津 300211
- Keywords:
Mesenchymal stem cells;
Osteoporosis;
Wnt signaling pathway;
miR-485-5p
- From:
Chinese Journal of Orthopaedics
2024;44(16):1104-1113
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To explore the role and mechanism of miR-485-5p targeted regulation of WNT7B in regulating osteogenic differentiation of bone marrow mesenchymal stem cell (BMSC). Methods:15 osteoporotic patients who underwent hip replacement due to hip fracture in Tianjin Hospital from January to October 2023 were collected, and bone tissues in the femoral head in the area of reduced bone density detected by the dual-energy X-ray absorptiometry (DXA) method were collected (osteoporosis group); 15 patients who underwent joint replacement due to osteoarthritis were matched according to their age and body mass index, and bone tissues in the femoral head in the area of normal bone density were collected (no osteoporosis group). MiR-485-5p and WNT7B were detected using qRT-PCR technology; the target genes and potential mechanisms of miR-485-5p were predicted using bioinformatics technology, and the relationship between miR-485-5p and WNT7B was analyzed by dual luciferase reporter system. The miR-485-5p overexpression (mimic) and inhibitor (inhibitor) were constructed and divided into control, miR-485-5p group and miR-485-5p inhibitor group. After alkaline phosphatase staining (ALP) and alizarin red staining (ARS), osteogenesis-related proteins were detected by Western blot (ALP, BMP-2, Runx2, OPN, OCN); expression of osteogenic proteins was detected by transfection of miR-485-5p inhibitor and WNT7B siRNA into BMSC. Results:The relative expression of miR-485-5p in the osteoporosis group was 7.54±0.49, which was higher than that in the no-osteoporosis group with significant difference ( t=4.11, P<0.001), while the relative expression of WNT7B was significantly lower ( t=3.38, P<0.001), which was negatively correlated with miR-485-5p; bioinformatics analysis found that miR-485-5p targeted 666 genes, miR-485-5p could bind the 3'UTR of WNT7B, and the main mechanism was related to the Wnt/β-catenin signaling pathway; ALP activity and calcium deposition were reduced in the miR-485-5p group compared with the control group, and ALP, BMP-2, Runx2, OPN, OCN, WNT7B and β-catenin proteins were 0.78±0.13, 0.68±0.16, 0.59±0.19, 0.54±0.14, 0.74±0.12, 0.49±0.17, 0.52±0.19, respectively, which were significantly reduced compared with the control group ( t=3.214, P<0.001; t=3.637, P<0.001; t=3.479, P<0.001; t=4.062, P<0.001; t=4.271, P<0.001; t=4.164, P<0.001; t=4.621, P<0.001), and ALP activity, calcium deposition were reduced; ALP, BMP-2, Runx2, OPN, OCN in miR-485-5p inhibition group, WNT7B and β-catenin protein relative expression were 1.29±0.21, 1.24±0.19, 1.16±0.24, 1.31±0.27, 1.45±0.25, 1.05±0.19, 1.41±0.26, respectively, which were significantly higher compared with the control group ( t=3.156, P<0.001; t=3.645, P<0.001; t=3.473, P<0.001; t=3.954, P<0.001; t=4.006, P<0.001; t=3.889, P<0.001; t=4.513, P<0.001). The relative expression of OPN, WNT7B and β-catenin proteins in the miR-485-5p inhibition group were 1.42±0.21, 1.38±0.32, 1.16±0.2.ALP activity was significantly lower in the miR-485-5p inhibition+WNT7Bi group, with lighter ARS staining, fewer bone deposits, and reduced bone-forming related proteins OPN, WNT7B and β-catenin relative expression of 1.08±0.19, 0.71±0.22, and 0.84±0.25, which were all significantly reduced ( t=3.675, P<0.001; t=3.401, P<0.001; t=3.354, P<0.001). Conclusion:MiR-485-5p overexpression slowed down the process of osteogenic differentiation and caused down-regulation of the expression of related proteins, whereas miR-485-5p inhibition promoted osteogenic differentiation and was negatively correlated with WNT7B in the bone tissues of osteoporosis patients. MiR-485-5p binds to the WNT7B mRNA target, which in turn influences the expression of related proteins of WNT7B, and the mechanism of its action is that miR-485-5p targeted to regulate WNT7B-mediated Wnt/β-catenin signalling pathway inhibits BMSC osteogenic differentiation.
