Study On The Mechanism Of Long Non-coding RNA Regulation Of MiR-150-5p/MMP In Intervertebral Disc Degenerative Diseases

BackgroundLow back pain(LBP) is a common disorder met in the spine department which affects normal daily life at different extents.Intervertebral disc degeneration(IDD) is one of the leading causes of LBP.However,its etiology and pathogenesis remain elusive which attributes to multi-factorial impacts.A growing number of evidences have indicted important roles of cellular and molecular abnormalities played in IDD among which expression abnormalities concerning non-coding RNAs that bear regulatory functions have gained a wide attention.Previous study has reported down regulation of miR-150-5p in IDD samples.We have predicted binding sites between miR-150-5p and long non-coding RNA(lncRNA)PART1 and LINC01121.Therefore,this study will focus on the mechanism of the above-mentioned two non-coding RNAs in regulation of mi RNA and their roles played in the degeneration of human nucleus pulposus(NP)cells.MethodsFirst,we had collected degenerated and normal human NP cell specimens.Second,quantitative real-time PCR(qRT-PCR)was used to determine the expression differences of lncRNA PART1,LINC01121 and miR-150-5p between two types of samples and then analyzed their correlation with clinical degenerative severity.Furthermore,NP cells were induced with different concentrations of IL-6 and IL-1β and the effects of inflammatory mediators on the expression of the above three subjects were measured.The lipofectamine 2000 method was used to construct the models of overexpression of the three designated RNAs in NP cells.Furthermore,their effects on nucleus pulposus cell proliferation were evaluated.The qRT-PCR technology was used to evaluate the regulation of miR-150-5p expression by LINC01121 and PART1.Luciferase reporter gene was used to study the effect of miR-150-5p on the expression of wild-type and mutant-type MMP-16.Finally,the effects of MMP-16 expression on nucleus pulposus cell proliferation(Ki-67),extracellular matrix degradation(MMP-3,ADAMTS5,type II collagen)expressions and the level of inflammatory mediators(IL-6,IL-1β,TNF-α)secretions in the culture medium were analyzed.ResultsWe have first confirmed that LINC01121 and PART1 were highly expressed in human degenerated NP cells and were positively correlated with the severity of disc degeneration.At the same time,miR-150-5p showed low expression in degenerated NP cells.The overexpression of the above two lncRNAs inhibited the expression of miR-150-5p,while the low expression of miR-150-5p promoted the up-regulation of mutant MMP-16.Consequently,it inhibited the expression of type Ⅱ collagen,enhanced the expression of MMP-3 and ADAMTS5 which related to the degradation of extracellular matrix,promoted the abnormal proliferation of NP cells and increased secretion of the inflammatory mediators IL-6,IL-1β,TNF-α.ConclusionsLINC01121 and PART1 are highly expressed in human degenerative disc nucleus pulposus cells and tissues and are correlated with degenerative severity.The above two lncRNAs mediate miR-150-5p/MMP-16 axis by promoting abnormal proliferation of NP cells,abnormal extracellular matrix degradation,increased inflammatory mediators secretions.All these factors are believed to be involved in the pathogenesis and development of degenerative changes of the lumbar spine.It is expected to become the target of diagnosis and treatment in the future.