Study On Mechanism Of LncRNA MALAT1 And NEAT1 In The Process Of Ossification Of The Posterior Longitudinal Ligament Caused By Mechanical Stimulation

OBJECTIVE Ossification of the posterior longitudinal ligament(OPLL)is a common spinal disease,especially in the Asian population,which is one of the major diseases that seriously endangers patient’s health.Our group had found that mechanical factors play an essential role for the ossification of posterior longitudinal ligament,and successfully established the posterior longitudinal ligament fibroblast cells osteogenic differentiation model induced by mechanical stretch in vitro.Although the previous study found some interpretation on the ossification of the posterior longitudinal ligament caused by mechanical stretch,but still can not explain the exact mechanism,and need to further explore.Long non-coding RNA(lncRNA)is a non-coding RNA with length greater than 200 nt,which can regulate the expression of genes at multiple levels.There had no report on the mechanism of lncRNA in ossification of posterior longitudinal ligament.Therefore,this study will explore the role and mechanism of lncRNA in the process of ossification of the posterior longitudinal ligament caused by mechanical stimulation.METHOD The total RNA was extracted from the ossification and non-ossified tissue of the posterior longitudinal ligament of the cervical spine,and the expression of lncRNA in the ossification and non-ossified tissue of the posterior longitudinal ligament of the cervical spine was detected though lncRNA microarray.The differential expression of lncRNA was verified by real-time quantitative PCR.The posterior longitudinal ligamentic fibroblast cells were isolated and the mechanical stretch was performed to detect the changes of lncRNA expression.Analysis of lncRNA-bound miRNAs using the starbase assay software,and then using Targetscan software to analyze mi RNA target genes.The expression of lncRNA in ligament fibroblast cells was regulated by siRNA,and then the expression of miRNAs and target genes was detected.The expression of miRNAs in ligament fibroblast cells was regulated and miRNA luciferase reporter vector was constructed to verify the target gene of miRNA.RESULT The results of lncRNA microarray showed that the expression of lncRNA in ossification and non-ossification tissue of posterior longitudinal ligament was different.The expression of lncRNA MALAT1 and lncRNA NEAT1 in ossification of posterior longitudinal ligament of cervical spine was significantly higher than that in non-ossified tissue.The mechanical stretch stimulation could significantly promote the expression of lncRNA MALAT1 and NEAT1 in cervical posterior longitudinal ligament fibroblast cells.The starbase analysis result showed that lncRNA MALAT1 could bind to mi R-30a-5p.The Targetscan software analysis showed thet miR-30a-5p had the target gene RUNX2.Mechanical stretch stimulates the expression of lncRNA MALAT1 in ligament fibroblast cells,and decreases the expression of miR-30-5p to promote the expression of target gene RUNX2.CONCLUSION The expression of lncRNA in the posterior longitudinal ligament ossification and non-ossification tissue was different.Mechanical stretch stimulation promoted the expression of lncRNA MALAT1 and NEAT1 in fibroblast cells of posterior longitudinal ligament.LncRNA MALAT1 promoted the expression of RUNX2 by regulating the expression of miR-30-5p.