The miR-623/CXCL12 axis inhibits LPS-induced nucleus pulposus cell apoptosis and senescence
Introduction
Intervertebral disc degeneration (IDD) is a chronic degenerative disease characterized by intracellular and biochemical changes in intervertebral discs cells, leading to structural deterioration of intervertebral discs (Deyo et al., 1990; Vergroesen et al., 2015). Notably, about 90 % of people above age 50 have IDD, suggesting that IDD increases with the growth of age (Cheung et al., 2009).
The components of intervertebral disc include NP (nucleus pulposus), AF (annulus fibrosus) and CEP (cartilaginous endplate). As the most hydrated region of intervertebral disc, NP enables intervertebral disc to absorb compressive forces and maintain the physiological function of discs through NP cells (NPCs)-secreted extracellular matrix (ECM) (Le Maitre et al., 2007a), including Collagen I, Collagen II, and proteoglycan (Adams and Roughley, 2006). However, with age, excessive apoptosis and senescence of NPCs are frequently observed in intervertebral disc. The senescence-associated NPC secretion, which consists of various cytokines, chemokines and matrix proteases, promotes ECM degradation and induces induce adjacent NPC death or senescence (Feng et al., 2016; Podichetty, 2007; Zhao et al., 2007a). Therefore, senescent NPCs lead to a defect in intervertebral disc homeostasis, inducing and/or aggravating IDD (Walker and Anderson, 2004; Gopal et al., 2012). Thus, targeting the apoptosis and senescence of NPCs might be a promising strategy for IDD treatment (Ding et al., 2013; Rannou et al., 2004; Le Maitre et al., 2007b; Roberts et al., 2006a).
As we have mentioned, the senescence-associated NPC secretion consists of various cytokines, chemokines and matrix proteases. During IDD, proinflammatory mediator levels are increase, whereas aggrecan and collagen degradation are enhanced (Roberts et al., 2006b). With the development of IDD, NPCs secret more proinflammatory molecules, including TNF-α, IL‑1α, IL‑1β, IL‑6, IL‑17, IL‑8, IL‑2, IL‑4, IL‑10, interferon‑γ (IFN‑γ) and so on (Le Maitre et al., 2005, 2007c), initiating and/or promoting the senescence or apoptosis of NPCs (Roberts et al., 2006b; Purmessur et al., 2013; Shen et al., 2011). Moreover, TNF-α and IL‑1β also elicit the levels of enzymes that degrade ECM, including matrix metalloproteinase (MMP)‑1, 3, 7, 9, 13 (Wang et al., 2011a; Le Maitre et al., 2007d; Pockert et al., 2009; Bachmeier et al., 2009), further enhancing the degradation of ECM and aggravating IDD. Inhibiting the overproduction of these pro-inflammatory factors may help to improve IDD.
Both genetic and environmental factors contribute to IDD, a multifaceted spinal disease (Battie et al., 2009, 2008). Over the past few years, a series of newly identified ncRNAs (noncoding RNAs), particularly miRNAs (microRNAs), have been reported participating in the pathogenesis of many diseases. miRNAs directly interact with AGO (Argonaute) protein, leading to inhibition of the expression of their downstream targets (Bartel, 2009). Through targeting different vital factors, miRNAs act on almost all the aspects of IDD pathogenesis (Li et al., 2015; Wang et al., 2015). For example, miR‐155 is one of the miRNAs proven to be implicated in regulating apoptotic pathways and immune responses (Wei et al., 2013), which represses the expression levels of FADD and caspase‐3 to inhibit NPC apoptotic processes (Wang et al., 2011b). Protein kinase C activation induced miR‐377 up‐regulation of miR‐377, followed by the decreases within ADAMTS5 and cleaved aggrecan (Tsirimonaki et al., 2013). With the development of high throughput sequencing technique, more and more miRNAs have been reported to be differentially expressed in IDD (Guo et al., 2017; Zhao et al., 2014); thus, identifying miRNAs acting on NPC apoptosis, senescence, and secretion of inflammatory factors and ECM degradation might provide novel targets for IDD treatment.
In the present study, we analyzed GSE63492 and GSE116726 for miRNAs differentially expressed between normal and IDD tissues and identified miR-623. miR-623 expression showed to be validated within collected clinical tissue specimens and LPS-stimulated and non-stimulated NPCs. Then, the specific effects of miR-623 on NPC proliferation, apoptosis, senescence, secretion of inflammatory factors, and ECM levels were examined under LPS stimulation. Candidate downstream targets of miR-623 were analyzed and applied for KEGG and GO signaling enrichment analyses; CXCL12 was chosen because of the role in human degenerative NPC apoptosis, inflammation, and matrix degradation (Liu et al., 2016, 2018a). The predicted binding between miR-623 and CXCL12 was validated and the effects of CXCL12 on LPS-stimulated NPCs were detected. Finally, the dynamic effects of the miR-623/CXCL12 axis upon CXCL12 expression and LPS-stimulated NPCs were examined to investigate whether miR-623 exerted its effects through CXCL12.
Section snippets
Clinical tissue samples
Twelve normal lumbar disc specimens and twelve degenerative specimens were collected as surgical waste from individuals undergoing elective spinal surgical procedures with the approval of Institutional Review Board guidelines of the Yiyang Central Hospital. The informed consent was signed by each patient enrolled.
NPC isolation and identification
NPC cells were isolated from 10 IDD patients’ NP tissues as described before (Wang et al., 2018a). Generally, NP tissues were cut into 1 mm3 and treated with 0.1 % collagenase and 10
miR-623 expression based on bioinformatics and experimental analyses
To identify miRNAs differentially expressed in normal and IDD tissue samples, we downloaded and analyzed two datasets from Gene Expression Omnibus (GEO), GSE63492 and GSE116726, and compared downregulated and upregulated miRNAs in IDD tissues. As shown in Table 1, these two datasets intersected at 21 miRNAs (14 upregulated and 7 downregulated). Among 7 downregulated miRNAs, the role of miR-486-3p (Cui and Zhang, 2020), miR-155 (Qu et al., 2017; Ye et al., 2016), and miR-486-5p (Chai et al., 2019
Discussion
Herein, miR-623 showed to be downregulated within IDD tissue samples according to both bioinformatics and experimental analyses. In LPS-injured NPCs, miR-623 overexpression promoted LPS-suppressed cell proliferation; moreover, miR-623 overexpression inhibited cell apoptosis and senescence, increased ECM secretion, and reduced levels of inflammatory factors. In contrast to miR-623, CXCL12 expression was significantly upregulated in IDD tissues; miR-623 directly bound CXCL12 to inhibit its
Acknowledgement
This study was supported by Natural Science Foundation of Hunan Province, China (2018JJ2410).
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