LncRNA TRPC7-AS1 regulates nucleus pulposus cellular senescence and ECM synthesis via competing with HPN for miR-4769-5p binding
Introduction
Intervertebral disc (IVD) degeneration (IDD) is considered as a chronic, multifactorial disc disease. The decreased proteoglycan content of NP (nucleus pulposus) and the imbalance of ECM (extracellular matrix) synthesis and degradation appear to be the main events on IDD pathophysiology (da Silva et al., 2009). An in-depth understanding of the dysregulation mechanisms of genes encoding these proteins may contribute to targeted therapy of IDD.
Human healthy IVDs consist of the peripheral AF (annulus fibrosus), the central NP (nucleus pulposus), and the upper and lower cartilaginous endplates. The NP (nucleus pulposus) is composed of NPCs (NP cells) that exert an essential effect on generating and maintaining IVD matrix (Stemple, 2005). As the body ages, the NPCs tend to senescence, thus losing their capacity of proliferating and renewing necrosis or apoptosis (Roberts et al., 2006; Gruber et al., 2007). Reportedly, up to 92 % of IVD cells were SA-β-Gal-positive during aging among middle-aged patients following IVD surgery (Gruber et al., 2007). Investigating the NPCs dysfunction mechanism may provide a new option for IDD treatment.
IDD is a multi-factor spinal disease that could be jointly regulated by genetic factors and environmental factors, mainly affected by genetic factors (Battie et al., 2009, 2008). The genetic mechanism ranges from single nucleotide variants (Song et al., 2013) and coding genes (Gruber et al., 2011, 2012; Sun et al., 2013a, b) to newly defined ncRNAs (noncoding RNAs), including miRNAs (small ncRNAs) and lncRNAs (long non-coding RNAs). miRNAs inhibit the expression of their targets via directly acting on an effector protein AGO (Argonaute) (Bartel, 2009); whereas lncRNAs interact with miRNAs through miRNA sponges and ceRNA (competing endogenous RNA) to play a role in post-transcriptional regulation (Paraskevopoulou and Hatzigeorgiou, 2016; Jalali et al., 2013). Previous studies have reported that a huge number of mRNAs, miRNAs and lncRNAs are dysregulated in IDD (Liu et al., 2015; Wan et al., 2014), indicating that lncRNA-miRNA-mRNA network might involve in IDD progression.
Herein, we downloaded non-coding RNA profiling (GSE56081 (Wan et al., 2014) and GSE63492 (Liu et al., 2015)) and performed GO annotation and enrichment analysis and association analyses on differentially-expressed genes to construct a lncRNA-miRNA-mRNA network that can regulate NPC proliferation, senescence and ECM synthesis in IDD. After that, the functions and correlations of the components were investigated. In summary, we regard lncRNA-miRNA-mRNA modulation as a new potent target for IDD treatment.
Section snippets
Clinical tissue samples
Eighteen normal lumbar disc and twenty-six degenerative specimens were collected as surgical waste from individuals undergoing elective spinal surgical procedures with the approval of Institutional Review Board guidelines of The Second Xiangya Hospital, Central South University. The informed consent was signed by each patient enrolled.
Cell line, cell culture and cell transfection
NPC cells were obtained from our previous study (Wang et al., 2018) and cultured as previously described. Briefly, For NPC isolation, about 200 g NP tissue from
Construction of lncRNA-miRNA-mRNA network associated with ECM during IDD
To identify possible components of lncRNA-miRNA-mRNA network associated with ECM during IDD, we downloaded microarray analyses results (GSE56081 (Wan et al., 2014) and GSE63492 (Liu et al., 2015)) and performed cluster analyses on these online data. Based on GSE56081 and GSE63492, differentially expressed mRNAs (fold change ≥2, p < 0.05), miRNAs (fold change ≥2, p < 0.05), and lncRNAs (fold change ≥2, p < 0.05) between the IDD nucleus pulposus tissues and normal nucleus pulposus tissues were
Discussion
Herein, we found that lncRNA TRPC7-AS1, miR-4769−5p, and HPN form a network in NPC via direct targeting to modulate NPC viability, senescence and ECM synthesis. TRPC7-AS1 and HPN expression are significantly upregulated while miR-4769−5p expression is downregulated in IDD tissue samples, indicating that lncRNA TRPC7-AS1/miR-4769−5p/HPN may be a promising target for IDD treatment.
IDD is regarded as a multi-factor spinal disease that could be jointly regulated by genetic factors and environmental
Acknowledgments
This study was supported by the National Natural Science Foundation of China (Grant No. 81802211 and 81871821) and the Natural Science Foundation of Hunan Province (Grant No. 2019JJ40412)
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The authors contributed equally to this study