microRNA-25-3p suppresses osteogenic differentiation of BMSCs in patients with osteoporosis by targeting ITGB3

https://doi.org/10.1016/j.acthis.2022.151926Get rights and content

Highlights

  • BMSCs from OP patients had highly expressed miR-25–3p and lowly expressed ITGB3.

  • miR-25–3p inhibited osteogenic differentiation of BMSCs from OP patients.

  • ITGB3 promotes osteogenic differentiation of BMSCs from OP patients.

  • miR-25–3p targeted to inhibit ITGB3.

  • miR-25–3p/ITGB3 axis inhibits osteogenic differentiation of BMSCs in OP patients.

Abstract

This study was conducted to investigate the impact of the microRNA (miR)-25–3p/ITGB3 axis on the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) from patients with osteoporosis (OP). BMSCs isolated from the bone marrow of healthy controls and OP patients were identified by flow cytometry, in which ITGB3 mRNA and miR-25–3p expression was detected by RT-qPCR and ITGB3, Runx2, OPN, ALP, and OSX protein expression by western blot. The binding between ITGB3 and miR-25–3p was assessed by dual-luciferase reporter gene and Ago2-RIP assays. BMSC osteogenic differentiation was observed by alizarin red staining and ALP activity. The differentiation of BMSCs to adipocytes and chondrocytes was measured by oil red O staining and alcian blue staining, respectively. BMSCs were successfully isolated from the bone marrow of healthy controls (normal-BMSCs) and OP patients (OP-BMSCs). ITGB3, Runx2, OPN, ALP, and OSX expression was poorer and miR-25–3p expression was higher in OP-BMSCs than in normal-BMSCs. Mechanistically, ITGB3 was negatively targeted by miR-25–3p. After osteogenic, adipogenic, and chondrogenic differentiation of BMSCs were successfully induced, adipogenic differentiation increased and osteogenic and chondrogenic differentiation decreased in OP-BMSCs compared with normal-BMSCs. Overexpression of ITGB3 facilitated mineralized nodule formation and elevated ALP activity and Runx2, OPN, and ALP expression in OP-BMSCs. miR-25–3p upregulation diminished mineralized nodule formation, ALP activity, and Runx2, OPN, and ALP expression in OP-BMSCs and normal-BMSCs, which was annulled by additional ITGB3 overexpression. miR-25–3p targets ITGB3, thereby suppressing osteogenic differentiation of BMSCs from OP patients.

Introduction

Osteoporosis (OP) is the most common disease of bone metabolism with a very high prevalence, and women are usually at higher risk than men (Ensrud and Crandall, 2017). OP is typified by low bone mass and microarchitectural degeneration of bone tissues, leading to increased bone frangibility and subsequently elevated danger of fracture (1993). Age is one of the most important factors influencing the incidence of fractures caused by OP (Ensrud and Crandall, 2017). OP and fractures associated with OP are common causes of morbidity and mortality in the elderly (Johnston and Dagar, 2020). OP is also considered a silent disease because there is usually no symptom until the first fracture occurs (Johnston and Dagar, 2020). In fact, OP is caused by an imbalance between bone resorption by osteoclasts and bone formation by osteoblasts (Wang et al., 2021). The most basic and effective measure to treat OP is to reduce bone resorption and increase bone formation (Wang et al., 2021). It has been proposed that microRNAs (miRNAs) affect the osteogenic differentiation process of bone marrow mesenchymal stem cells (BMSCs) (Wang et al., 2019).

miRNAs, a class of small non-coding RNAs, can bind to target genes to participate in various biological processes, including cell propagation, differentiation, and apoptosis, as well as organ development (Wang et al., 2021). Liu et al. identified miR-25–3p as a repressor of osteogenesis in human umbilical cord stem cells (Cao et al., 2020). miR-25–3p is a suitable reference gene for OP induced by estrogen deficiency and mechanical unloading because miR-25–3p was stably expressed in OP or non-OP serum and bone tissues (Chen et al., 2016). On contrary, evidence in a previous study also showed that miR-25–3p regulated the expression of NFIX to facilitate osteoclast activity (Huang et al., 2020). Considering the controversial role of miR-25–3p in osteogenic differentiation, this study aims to determine whether miR-25–3p has a certain role in osteogenic differentiation of BMSCs.

A study has suggested that integrin-beta3 (ITGB3) is an osteoblast-specific gene (Asagiri and Takayanagi, 2007). It has been demonstrated that IL1RN interacts with ITGB3 to activate β-linked protein signaling, which ultimately promotes osteoblast differentiation (Zou et al., 2021), indicating the possible implication of ITGB3 in osteoblast differentiation. The binding sites between miR-25–3p and ITGB3 were predicted by starBase. We, therefore, determined the possible effect of the miR-25–3p/ITGB3 axis on BMSC osteogenic differentiation under the context of OP.

Section snippets

Sample collection

Bone marrow tissues were collected from six patients [aged (53 ± 6.92) years, including 4 females and 2 males] diagnosed with OP who underwent hip surgery at the First Hospital of Changsha, and normal bone marrow tissues donated by three injured patients [aged (50 ± 4.85) years, including 2 females and 1 male] were also harvested. Patients with gout, rheumatoid arthritis, systemic inflammatory diseases, autoimmune diseases, or chronic malignant diseases were excluded from this study. Written

Osteogenic differentiation of BMSCs was reduced in OP patients

To identify whether cells isolated from bone marrow were BMSCs, the expression of BMSC biomarkers (CD73, CD90, CD105, CD34, and CD45) was measured by flow cytometry. CD73, CD90, and CD105 were utilized as positive markers for BMSCs, and the hematopoietic stem cell marker CD34 and the leukocyte marker CD45 were used as negative markers. The results showed that the cells separated from bone marrow of normal controls (normal-BMSCs) and OP patients (OP-BMSCs) had the positive expression of CD73,

Discussion

OP is a common disease that causes severe bone loss and may lead to pathological fractures, which can reduce activities of daily living and quality of life. Therefore, OP prevention needs to be taken seriously. In our research, miR-25–3p and ITGB3 expression was determined in BMSCs extracted from OP patients and normal individuals. The findings indicated that ITGB3 was lowly expressed and miR-25–3p was highly expressed in OP-BMSCs. Moreover, this study further demonstrated that miR-25–3p

Conclusion

In summary, our findings stated that the miR-25–3p/ITGB3 axis regulated osteogenic differentiation of BMSCs from OP patients. This study aims to provide some new theoretical and experimental basis for OP treatment. The limitation of this study is that only the impact of the miR-25–3p/ITGB3 axis on BMSC osteogenic differentiation was investigated. The upstream and downstream of this axis will be further investigated in the future to advance our understanding of the miR-25–3p/ITGB3 axis in BMSC

Author statement

No conflict of interest exits in the submission of this manuscript, and manuscript is approved by all authors for publication. I would like to declare on behalf of my co-authors that the work described was original research that has not been published previously, and not under consideration for publication elsewhere, in whole or in part. All the authors listed have approved the manuscript that is enclosed.

Funding

This study was supported by Projects of Health and Family Planning Commission of Jiangxi Provincial (No.20177013; No.202140022), Changsha Science and Technology Bureau (No.kq2004157) and Hunan Provincial Health Commission (No.202201042974).

Authors’ contributions

YDP conceived the ideas. YDP and LZ designed the experiments. YDP, LZ and CJ performed the experiments. YDP and SF analyzed the data. YDP, LZ and WGW provided critical materials. YDP and LZ wrote the manuscript. YDP supervised the study. All the authors have read and approved the final version for publication.

Competing interest

The authors declare there is no conflict of interest regarding the publication of this paper.

Acknowledgment

We acknowledge Guowen Wei (Department of Orthopedics, The First Hospital of Nanchang, Nanchang, China) for her valuable advice. We acknowledge Zhen Li (Department of Pathology, The First Hospital of Changsha City, Changsha, China) for her support.

References (22)

  • K.E. Ensrud et al.

    Osteoporosis

    Ann. Intern. Med.

    (2017)
  • Cited by (6)

    1

    Dongping Yu and Zhen Li contributed equally to this research.

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