By inhibiting ADCY5, miR-18a-3p promotes osteoporosis and possibly contributes to spinal fracture

https://doi.org/10.1016/j.bbrc.2021.02.118Get rights and content

Highlights

  • Over-expression of ADCY5 is involved in osteogenic differentiation.

  • ADCY5 is a direct target of miR-18a-3p.

  • MiR-18a-3p regulate osteogenic differentiation via ADCY5.

Abstract

To investigate the influence of miR-18a-3p and ADCY5 on OP and osteogenic differentiation of human Mesenchymal stem cell (hBMSCs) and its possible mechanism. Samples were collected from osteoporotic patients with or without vertebral compression fracture, and without OP volunteers. MiR-18a-3p and ADCY5 mRNA expression levels in the tissue samples and hBMSCs during osteogenic differentiation were detected。MiR-18a-3p mimic and OE-ADCY5 were introduced into hBMSCs to research the effects of miR-18a-3p and ADCY5 on osteogenesis differentiation of hBMSCs. Dual luciferase reporter system and RNA pull-down were applied to determine whether ADCY5 was a target gene of miR-18a-3p. Compared with the control group, ADCY5 expression level was down-regulated in patients with OP-no-Frx and OP-Frx, but that of miR-18a-3p was up-regulated. In addition, ADCY5 increased during osteogenesis differentiation of hBMSCs, whereas miR-18a-3p did not. OE-ADCY5 significantly facilitated calcium deposition, ALP activity, osteoblast protein expression (OSX, ALP and EUNX2), miR-18a-3p mimic inhibited osteogenic differentiation, and partially reversed the effect of OE-ADCY5 on osteogenic differentiation. In general, miR-18a-3p targets ADCY5 to promote OP and may be involved in spinal fracturs.

Introduction

Osteoporosis (OP), a chronic musculoskeletal disease induced by the destruction of bone microstructure, the increase of bone fragility and the decrease of bone mass [1]. The Pathophysiology of OP is the out of balance in bone remodeling as osteoclasts work more than osteoblasts [2]. The consequence of OP is an aggradation risk of fracture [3]. Bone remodeling relies on the equilibrium between osteoclast-mediated bone resorption and osteoblast-mediated bone formation, which is of great significance in the treatment of OP. The pathological process of OP is influenced by many factors such as hormones, genetic regulatory factors and living environment [4]. In the last few years, numerous studies have confirmed that MicroRNAs (miRNAs) take an indispensable part in the regulation of bone formation. MiRNAs are identified as a viable biomarker of disease diagnosis and prognosis, including OP [5].

MicroRNAs (MiRNAs), small non-coding RNA molecules. Mature miRNAs range in size from 21 to 23 nucleotides. The function of miRNA contains adjust the expression of target gene by post transcriptional silencing [6]. Due to its stability, easy access to patient samples, and disease specificity, miRNAs are considered to be a biomarker for OP [6,7]. For example, MiR-194–5p worsens OP by inhibiting osteogenic differentiation [8]. MiR-664a-5p accelerates the progression of osteogenic differentiation of hBMSCs [9]. However, there has been little research on miRNA expression in osteoporotic vertebral fractures. Previous reports have found that overexpression of miR-18a-3p may be involved in OP and peripheral and column fractures [10]. Therefore, we investigated the value of miR-18a-3p in osteogenic processes in OP with or without vertebral fracture.

Adenylate cyclase 5 (ADCY5) has the ability to bind to the adenylate cyclase family and convert adenosine triphosphate (ATP) into the second messenger cyclic adenosine monophosphate (cAMP) [11]. ADCY5 is probably the largest constituent of the ADCY family in the islets of human [12] and is involved in the morphological changes of the ovaries [13], Nocturnal dyskinesia [14].

Nevertheless, the effect of miR-18a-3p and ADCY5 in the differentiation of osteoblasts from OP has not been studied. hBMSCs have the capacity to differentiate into osteoblasts, chondroblast and myoblasts [15,16]. In this research, miR-18a-3p expression was detect in bone tissue. At the same time, we studied the influence of miR-18a-3p and ADCY5 on the differentiation of osteoblasts in OP, and discussed the possible molecular mechanism.

Section snippets

Human samples

Bone tissues were collected from osteoporotic patients with or without vertebral compression fractures or no OP volunteers in our hospital. This project has been approved by the Research Ethics Committee of our hospital. All patients were enrolled in the program with informed consent. The volunteers were divided into OP (T-score 2.5), osteoporotic vertebral compression fractures (T-score 2.5 for Brittle Fractures) and no OP patients (T-score 1.0). All patients underwent hip replacement surgery.

Over-expression of ADCY5 is involved in osteogenic differentiation

To make certain of the association of ADCY2, ADCY5, and GRIA1 with OP, we analyzed the levels of ADCY2, ADCY5, and GRIA1. To this end, we analyzed clinical samples by qRT-PCR. As shown in Fig. 1A, ADCY5 levels significantly down-regulated in OP-no-Frx and OP-Frx than in non-OP patients. Furthermore, compared with OP-no-Frx, OP-Frx had significantly lower levels of ADCY5. Next, we began to analyze hBMSCs, a cell line with the potential to differentiate into osteoblasts. To detect changes in the

Discussion

A variety of miRNAs have been found to affect the expression of genes related to bone formation at the post-transcriptional level [17]. For instance, Bin Li et al. [18] found that 12 miRNAs, including miR-18a-3p, are excellent predictors of osteoarthritis, and are down regulated in osteoarthritis. Ding B et al. [19] reported that miR-18a-3p participates in chondrocyte apoptosis. However, it is not clear whether miR-18a-3p is directly concerned with the regulation of bone formation. This

Declaration of competing interest

The authors declare that they have no conflict of interests.

Acknowledgements

Not applicable.

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    The authors contributed equally to this work.

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