MicroRNA-494 promotes apoptosis and extracellular matrix degradation in degenerative human nucleus pulposus cells.

Liang Kang,Yu Song,Kun Wang,Yukun Zhang,Cao Yang,Kangcheng Zhao,Wei Liu,Wenbin Hua,Shuai Li,Ji Tu,Huipeng Yin

doi:10.18632/oncotarget.15838

Abstract

PurposeThis study investigated the expression and function of the microRNA-494 in intervertebral disc degeneration (IDD).ResultsMicroRNA-494 expression was upregulated during IDD progression; its overexpression increased the expression of ECM catabolic factors such as matrix metalloproteinase and A disintegrin and metalloproteinase with thrombospondin motif in NP cells while decreasing that of anabolic genes such as type II collagen and aggrecan; it also induced the apoptosis of NP cells, as determined by flow cytometry. These effects were reversed by microRNA-494 inhibitor treatment. SOX9 was identified as a target of negative regulation by microRNA-494. Promoter hypomethylation and NF-κB activation were associated with microRNA-494 upregulation in IDD.Materials and MethodsMicroRNA-494 expression in degenerative nucleus pulposus (NP) tissue was assessed by quantitative real-time PCR. The effect of microRNA-494 on extracellular matrix (ECM) metabolism and NP cell apoptosis was evaluated by transfection of microRNA-494 mimic or inhibitor. The regulation of SRY-related high mobility group box (SOX)9 expression by microRNA-494 was assessed with the luciferase reporter assay, and the methylation status of the microRNA-494 promoter was evaluated by methylation-specific PCR and bisulfite sequencing PCR. The role of activated nuclear factor (NF)-κB in the regulation of microRNA-494 expression was evaluated using specific inhibitors.ConclusionsMicroRNA-494 promotes ECM degradation and apoptosis of degenerative human NP cells by directly targeting SOX9.

Highlights

MicroRNA-494 expression was upregulated during Intervertebral disc degeneration (IDD) progression; its overexpression increased the expression of extracellular matrix (ECM) catabolic factors such as matrix metalloproteinase and A disintegrin and metalloproteinase with thrombospondin motif in nucleus pulposus (NP) cells while decreasing that of anabolic genes such as type II collagen and aggrecan; it induced the apoptosis of NP cells, as determined by flow cytometry
To investigate the effect of miR-494 on degenerative human NP cells, miR-494 mimic, miR-494 inhibitor, or miR-Scr were transfected into the cells and the expression of ECM anabolic genes was assessed by quantitative real-time (qRT)-PCR and western blotting
ECM catabolic proteinases such as matrix metalloproteinase (MMP) and A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTSs) are highly expressed in degenerative intervertebral disc tissue and cells, and have been linked to ECM degradation and IDD progression [25]; we evaluated the expression of MMP3, MMP13, ADAMTS4, and ADAMTS5 in NP cells

Summary

Introduction

Chronic low back pain (CLBP) is a common musculoskeletal disorder affecting up to 80% of individuals at some point during their lifetime, placing an economic burden on society [1, 2]. Intervertebral disc degeneration (IDD) is the major cause of CLBP [3]. Intervertebral discs are composed of the nucleus pulposus (NP), annulus fibrosus (AF), and cartilage end plates. NP cells maintain the homeostasis of the extracellular matrix (ECM), which includes type II collagen and aggrecan [6]. Excessive apoptosis of NP cells is an initiating event in IDD [7]. Decreases in type II collagen and aggrecan levels— which arise due to an imbalance between ECM anabolism and catabolism—and consequent ECM degradation are features of IDD [8].

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