LncRNA TRPC7-AS1 regulates nucleus pulposus cellular senescence and ECM synthesis via competing with HPN for miR-4769-5p binding

doi:10.1016/j.mad.2020.111293

Mechanisms of Ageing and Development

Volume 190, September 2020, 111293

https://doi.org/10.1016/j.mad.2020.111293 Get rights and content

Highlights

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lncRNA TRPC7-AS1, miR-4769−5p, and HPN form an axis to modulate the ECM in NPCs.
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miR-4769−5p target HPN 3'UTR to modulate HPN expression.
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TRPC7-AS1 service as a ceRNA to sponging miR-4769−5p, thus regulating NPC viability, senescence, and ECM synthesis.

Abstract

Intervertebral disc (IVD) degeneration (IDD) is identified as an abnormal, cell-mediated, age-dependent and genetics-dependent molecular degeneration process in which NPCs (nucleus pulposus cells) senesce and the balance of ECM (extracellular matrix) synthesis and catabolism is disrupted. Increasing evidence reveals that IDD can be modulated by genetic factors, including non-coding RNAs. In the present study, we downloaded non-coding RNA profiling (GSE56081 and GSE63492) and performed GO annotation and enrichment analysis and association analyses on differentially-expressed genes. LncRNA TRPC7-AS1, miR-4769−5p, and Hepsin (HPN) may form a lncRNA-miRNA-mRNA network that can regulate NPC proliferation, senescence and ECM in IDD. LncRNA TRPC7-AS1 directly targets miR-4769−5p while miR-4769−5p directly targets HPN 3′UTR. miR-4769−5p overexpression inhibited HPN expression, suppressed NPC senescence, promoted NPC viability, and promoted ECM synthesis. The effect of TRPC7-AS1 silence on NPCs was similar as miR-4769−5p overexpression while the effect of HPN overexpression was opposite to miR-4769−5p overexpression. miR-4769−5p suppression or HPN overexpression could significantly attenuate the effect of TRPC7-AS1 silence. LncRNA TRPC7-AS1 relieves miR-4769−5p-induced inhibition on HPN via acting as a ceRNA, thus regulating NPC viability, senescence, and ECM synthesis. In summary, we regard lncRNA-miRNA-mRNA modulation as a new potent target for IDD treatment.

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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¹: The authors contributed equally to this study

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LncRNA TRPC7-AS1 regulates nucleus pulposus cellular senescence and ECM synthesis via competing with HPN for miR-4769-5p binding

Highlights

Abstract

Introduction

Section snippets

Clinical tissue samples

Cell line, cell culture and cell transfection

Construction of lncRNA-miRNA-mRNA network associated with ECM during IDD

Discussion

Acknowledgments

Cell

Spine J.

J. Biol. Chem.

Trends Mol. Med.

Exp. Mol. Pathol.

J. Biol. Chem.

Genom. Data

Lab. Invest.

Int. J. Biochem. Cell Biol.

The cutting edge: membrane-anchored serine protease activities in the pericellular microenvironment

Biochem. J.

Genetic and environmental effects on disc degeneration by phenotype and spinal level: a multivariate twin study

Spine (Phila Pa 1976)

Senescence-like changes induced by expression of p21Waf1/Cip1 in NIH3T3 cell line

Cell Res.

lncRNAs: novel players in intervertebral disc degeneration and osteoarthritis

Cell Prolif.

[New perspectives on degenerative disease treatment]

Acta Reumatol. Port.

Senescence in cells of the aging and degenerating intervertebral disc: immunolocalization of senescence-associated beta-galactosidase in human and sand rat discs

Spine (Phila Pa 1976)

Genome-wide analysis of pain-, nerve- and neurotrophin -related gene expression in the degenerating human annulus

Mol. Pain

Gene expression profile identifies potential biomarkers for human intervertebral disc degeneration

Mol. Med. Rep.