Over-expression of miR-411–5p and miR-434–3p promotes the osteoblast differentiation by targeting GATA4

https://doi.org/10.1016/j.mce.2020.110759Get rights and content

Highlights

  • What's already known about this topic?

    • PIO is the main cause of aseptic loosening in patients after TJR surgery.

    • GATA4 plays an important role in osteoblast differentiation.

  • What does this study add?

    • The expression of miR-411–5p and miR-434–3p was decreased in osteolysis mice and UHMWPE-induced mMSCs.

    • MiR-411–5p and miR-434–3p targeted GATA4.

    • Overexpression of miR-411–5p and miR-434–3p promoted osteoblast differentiation through regulating GATA4.

Abstract

Objective

To investigate the role of miR-411–5p and miR-434–3p in osteoblast differentiation in particulate-induced osteolysis.

Methods

A mouse model of osteolysis and an in vitro osteolysis model were constructed. The expressions of molecules were detected using qRT-PCR and western blot. Alkaline phosphatase (ALP) activity was measured using the ALP Assay Kit, and the bone mineralization was measured using alizarin red staining.

Results

The expression of miR-411–5p and miR-434–3p was decreased in osteolysis mice and UHMWPE-induced mMSCs, while GATA4 protein expression was increased. Over-expression of miR-411–5p and miR-434–3p up-regulated the expressions of osteoblast gene markers, enhanced the ALP activity, promoted the bone mineralization of mesenchymal stem cells. In addition, miR-411–5p and miR-434–3p could target GATA4, and miR-411–5p/434–3p affected the expressions of osteoblast gene markers through GATA4 in vitro and in vivo.

Conclusion

Overexpression of miR-411–5p and miR-434–3p promoted the osteoblast differentiation by inhibiting GATA4 expression.

Introduction

Total joint replacement (TJR) is a common orthopaedic surgery for reducing joint pain in rheumatoid arthritis, osteoarthritis, and other end-stage joint diseases (Ru et al., 2016). Particulate-induced osteolysis (PIO) is the main cause of aseptic loosening in patients after TJR surgery, which affects the long-term stability and service life of artificial joints (Zhou et al., 2012). In the pathogenesis of PIO, wear particles stimulate the macrophages around the prosthesis to produce a variety of cytokines, which can disturb the differentiation, survival, and function of osteoblast and osteoclast, thus leading to an imbalance between bone formation and resorption (Deng et al., 2017). Although the effect of wear particles on osteoclasts and osteoblasts has been paid increasing attention, the mechanism of PIO is still lack of understanding.

MicroRNAs (miRNAs) are a class of non-coding small RNAs with a length of 20–24 nucleotides, which have a key role in the regulation of various biological processes (Ge et al., 2018). Recently, growing evidence reveals that multiple miRNAs are involved in osteoblast differentiation. Fukuda et al. (2015) reported that miR-145 could inhibit osteoblastic differentiation via Cbfb by forming a regulatory microRNA network. Liu et al. (2016) revealed that miR-9 promoted osteoblast differentiation of mesenchymal stem cells (MSCs) by suppressing DKK1 expression. MiR-411–5p, a lowly expressed miRNA in the tumor, can act as a tumor suppressor (Sun et al., 2015). MiR-434–3p, an anti-apoptotic miRNA, was reported by Pardo et al. (2017) in 2017. Based on the bioinformatics analysis by the UCSC database, we found that miR-411–5p and miR-434–3p are located on the same chromosome. It has been reported that miR-411–5p and miR-434–3p are down-regulated in MSCs and bone tissues from mice of age-related osteoporosis (He et al., 2013), implying that miR-411–5p and miR-434–3p might play an important role in the bone system. However, the role and mechanism of miR-411–5p and miR-434–3p in osteoblast differentiation has not been reported.

GATA family is a kind of transcriptional regulator with zinc-finger structure, which plays important roles in many biological processes (Shu et al., 2015). GATA4 is a member of the GATA family, which is mainly involved in cell proliferation, glycolysis, and inflammation (Kohlnhofer et al., 2016; Schrade et al., 2015; Kang et al., 2015). It has been found that GATA4 plays an important role in osteoblast differentiation (Song et al., 2014; Zhou et al., 2017; Guemes et al., 2014). Based on the results of the software prediction (TargrtScan), both miR-411–5p and miR-434–3p have binding sites of GATA4. Therefore, we speculated the role of miR-411–5p and miR-434–3p in osteoblast differentiation might be related to GATA4.

In this study, we investigated the underlying mechanism of miR-411–5p and miR-434–3p in osteoblast differentiation, aiming to provide a potential therapeutic target for PIO.

Section snippets

Wear particle preparation

Ultra-high molecular weight polyethylene (UHMWPE) particles (diameter ranges 0.1–10 μm) were purchased from Clariant (Frankfurt, Germany). To remove endotoxin, UHMWPE particles were washed twice in 75% ethanol for 24 h at room temperature. Then, UHMWPE particles were washed in phosphate-buffered saline (PBS) for three times and dried in a desiccator.

Animals

C57Bl/6 (wild-type, WT) mice were purchased from Guangdong Medical Laboratory Animal Center (Guangdong, China). All protocols were approved by the

The expressions of miR-411–5p and miR-434–3p were decreased in osteolysis mice and UHMWPE-induced mMSCs

In this part, we performed in vivo and in vitro experiments to test the relationship between miR-411–5p/434–3p and osteolysis. We established the mouse osteolysis model and measured the expressions of miR-411–5p/434–3p in the calvaria. As shown in Fig. 1A, the expressions of miR-411–5p and miR-434–3p were reduced in the UHMWPE group (i.e. osteolysis model group) compared with the sham group. In addition, GATA4 protein expression was increased in the UHMWPE group, while Runx2 protein expression

Discussions

The aseptic loosening caused by wear particles is still the main complication after TJR surgery. Previous studies have reported that wear particles can affect the differentiation, apoptosis, and metabolism of osteoblast, and then cause prosthesis loosening and osteolysis (Bu et al., 2017). Therefore, it is of great significance to study the effect of wear particles on osteoblasts to resolve PIO after TJR.

MiRNAs have been reported to be related to osteoblast differentiation (van Wijnen et al.,

CRediT authorship contribution statement

Xuren Gao: Formal analysis, Data curation, Conceptualization, Writing - original draft. Jian Ge: Data curation, Writing - review & editing. Weiyi Li: Data curation, Writing - review & editing. Wang-chen Zhou: Data curation, Writing - review & editing. Lei Xu: Formal analysis, Writing - review & editing. De-qin Geng: Formal analysis, Writing - review & editing.

Declaration of competing interest

All authors declare that they have no conflicts of interest in this work.

Acknowledgments

This study was supported by the Foundation of President of Xuzhou Medical University (No.2011KJZ05), the Foundation for the Returned Overseas Chinese Scholars of the Affiliated Hospital of Xuzhou Medical University (No. 2017), the Foundation of Xuzhou Science and Technology Bureau (No.KC18060) and the Post-doctoral Foundation of Jiangsu Province(No.2018412).

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