MicroRNA-186-5p Promotes The Extracellular Matrix Degeneration Of Nucleus Pulposus In Vitro Study

Objective: Intervertebral disc degeneration(IDD)is a very common orthopedic disease in the world.The incidence of IDD in people over the age of 60 in China can reach 90%,which can cause pain and herniation.Current treatments for IDD include conservative and invasive therapies.However,the anatomical structure of the intervertebral disc is inevitably changed during the surgical treatment.In addition,conservative treatment methods are limited to alleviating the symptoms caused by IDD,and cannot restore the structure and function of the intervertebral disc.Increasing evidence shows that micro RNAs(miRNAs)are a class of small non-coding RNAs.These small RNAs are related to the pathogenesis of many diseases,such as tumors,Alzheimer’s disease and osteoarthritis.In addition,a large number of studies have found that miRNAs are involved in regulating a variety of cellular biological processes,including apoptosis,proliferation,and aging.MicroRNAs silence protein expression by either inhibiting translation elongation or inducing mRNA decay when pairing with the 3 ’untranslated region(3’-UTR)of their target mRNA.Studies have shown that miRNA-186-5p(miR-186-5p)can improve the survival rate of chondrocytes,promote chondrocyte proliferation and inhibit chondrocyte apoptosis,and miR-186-5p may be the future of osteoarthritis treatment strategy.The purpose of this study was to study the expression of miRNA-186-5p in intervertebral disc degeneration and evaluate the effect of miRNA-186-5p on the extracellular matrix of the nucleus pulposus in degenerative discs,and to explore the possible mechanism of this process.Methods: Primary cells were obtained from tissue samples and cultured for subsequent experiments.MiR-186-5p mimic(miR-186-5p)and inhibitor(miR-186-5p-in)were transfected to achieve miR-186-5 overexpression and inhibition.Real-time fluorescence quantitative polymerase chain reaction(qRT-PCR)and Western blot were used to detect type II collagen,aggrecan,ADAMTS4 and matrix metalloproteinase 13(MMP-13).The dual luciferase reporter gene experiment was used to analyze whether miR-186-5p could bind to the potential target gene SIRT6.Results: Compared with non-degenerate nucleus pulposus,miR-186-5p was significantly up-regulated in degenerated nucleus pulposus of patients.In degenerate nucleus pulposus cells,overexpression of miR-186-5p leads to reduced expression levels of type II collagen and aggrecan and increased expression levels of ADAMTS and MMP-13;Down-regulation of miR-186-5p significantly increased the expression levels of type II collagen and aggrecan,and reduced the expression levels of ADAMTS and MMP-13.We also confirmed that SIRT6 is a direct target gene of miR-186-5p.Finally,the effect of increased expression of extracellular matrix components caused by suppressing the miR-186-5p can be reversed by SIRT6-specific interfering RNA(si-SIRT6).Conclusions: Our results show that miR-186-5p is significantly differentially expressed in degenerative nucleus pulposus tissue,suggesting that miR-186-5p may play an important role in the occurrence and progression of disc degeneration.We found that miR-186-5p can regulate the catabolism of the extracellular matrix to affect the intervertebral disc degeneration.Inhibition of miR-186-5p alleviates the degradation of extracellular matrix by increasing the expression of SIRT6,which clarifies the specific mechanism of miR-186-5p affecting the extracellular matrix of degenerated nucleus pulposus.Alleviating the degradation of the extracellular matrix of the nucleus pulposus by inhibiting the up-regulation of miR-186-5p expression may be a new strategy for the treatment and prevention of intervertebral disc degeneration,broadening the scope of our understanding of disease.