Exosomal miR-532–5p from bone marrow mesenchymal stem cells reduce intervertebral disc degeneration by targeting RASSF5

Highlights

• TNF-α reduces miR-532-5p expression in NPCs.

• TNF-α increases miR-532-5p expression in exosomes derived from BMSCs.

• BMSCs-Exos containing abundant miR-532-5p induce a better protective effect on the degenerated NPCs.

• MiR-532-5p inhibits NPC apoptosis, extracellular matrix degradation and fibrosis deposition to protect against IDD.

• RASSF5 is a direct target gene of miR-532-5p to promote NPC apoptosis.

Abstract

The transplantation of bone marrow mesenchymal stem cells (BMSCs) has been found to be used as an effective therapy of intervertebral disc degeneration (IDD). However, the underlying mechanisms of BMSCs in the progress of IDD are not fully explained. In this study, we found that exosomes derived from BMSCs (BMSCs-Exos) inhibited the apoptotic rate, extracellular matrix (ECM) degradation, and fibrosis deposition in TNF-α-induced nucleus pulposus cells (NPCs). Importantly, the level of miR-532–5p was observed to be decreased in apoptotic NPCs, but abundant in BMSCs-Exos with TNF-α treatment. The results showed that BMSCs-Exos under TNF-α stimuli exerted better effects on NPCs than BMSCs-Exos, which might be mitigated by the inhibition of miR-532–5p in BMSCs-Exos. The gain-of-function results suggested that the direct overexpression of miR-532–5p in NPCs could inhibit TNF-α-induced increase of apoptotic process, activation of apoptotic proteins, imbalance of anabolism/catabolism levels, and accumulation of collagen I. In addition, RASSF5 was demonstrated to be a target of miR-532–5p. Knockdown of RASSF5 could decrease the apoptotic cells and reduce the activated apoptotic protein levels in TNF-α-induced NPCs. Overall, these data indicate that exosomes from BMSCs may suppress TNF-α-induced apoptosis, ECM degradation, and fibrosis deposition in NPCs through the delivery of miR-532–5p via targeting RASSF5. This work provides a promising therapeutic strategy for the progress of IDD.

Introduction

Intervertebral disc degeneration (IDD) is considered as a main cause of chronic discogenic back pain and sciatica, leading to poor qualities of life in patients [1]. The intervertebral disc is composed of nucleus pulposus (NP), annulus fibrosus, and cartilage end-plates to maintain normal functions. It is reported that the nucleus pulposus cells (NPCs) play an essential role in the initiation of IDD [2]. As we known, the main characteristics of IDD are the loss of NPCs and the degradation of extracellular matrix (ECM) [3,4]. The high levels of inflammatory factors, like TNF-α, have been demonstrated to cause an excessive apoptosis of NPCs, thus resulting in IDD progression [5,6]. However, the exact mechanisms in IDD remain to be further elucidated.

Recently, increasing researchers make great focuses on the therapeutic effects of bone marrow mesenchymal stem cells (BMSCs) for IDD development due to the inhibitory capacity for NPC apoptosis [7,8]. Importantly, BMSCs are found to transfer exosomes to protect against the tissue or organ injury [9,10]. Exosomes are recognized as the double-membrane vesicles with a diameter of 40–120 nm, which are involved in cell communication and behavior by the delivery of bioactive molecules, including miRNAs, mRNAs and nutritional elements [11]. The transplantation of exosomes from BMSCs (BMSCs-Exos) into NPCs has been reported to inhibit the apoptosis of NPCs induced by inflammatory cytokines [[12], [13], [14]]. Thus, it is necessary for understanding the regulatory mechanisms of exosomes from BMSCs in the therapy of IDD.

MiRNAs are a class of small non-coding RNAs that regulate the target gene expression to mediate multiple biological processes, such as cell differentiation, apoptosis and angiogenesis [15,16]. Several studies have demonstrated the close implications of miRNAs in IDD development. For instance, Cheng et al. suggests that exosomal miR-21 could inhibit the apoptosis of NPCs by targeting PTEN [14]. Interestingly, the decreased expression of miR-532–5p is evidently observed in the NP tissues of IDD [17], implying its potential protection on IDD development. Accumulating reports suggest that miR-532–5p can inhibit the apoptosis of various cell types [18,19], which is found to be existed in the exosomes [20]. In addition, MMP-13, a regulator of ECM degradation in IDD, has been found to be complementary to miR-532–5p [[21], [22], [23]]. However, whether miR-532–5p derived from BMSCs participates in the degeneration of intervertebral disc is unclear. Considering that RASSF5, the pro-apoptotic gene, was predicted to directly target miR-532–5p, we speculated that miR-532–5p might inhibit NPC apoptosis by targeting RASSF5 to block ECM degradation and fibrotic deposition, protecting the development of IDD.