The Function And Mechanism Of LncRNA In The Regulation Of Nucleus Pulposus Cell Apoptosis During Intervertebral Disc Degeneration

[Objectives]Senescence and degeneration of intervertebral disc are the precondition and pathological basis of spinal degenerative disease,Understanding the biologic mechanisms of senescence and degeneration in the disc and exploiting of effective treatment and interventions for the health problem coming with intervertebral disc degeneration has become to be one of the global medical challenge currently and in the future.Through bioinformatics analysis,this study is carried out to screen of nucleus pulposus cell apoptosis related lncRNA and explore the mechanism of lncRNA in the regulation of nucleus pulposus cell apoptosis during intervertebral disc degeneration,which will provide scientific guidance for searching new therapeutic targets of degenerative disc disease.[Methods]Three bioinformatics analysis software were compared,and Target Scan was chosen to screen of lnc NRAs that may regulate nucleus pulposus cell apoptosis by targeting miR-155.Through miR-155 mimics transfection,expression of lncRNA in nucleus pulposus cells with miR-155 overexpression was detected by RT-PCR,then lncRNA and mRNA microarray was used to evaluate the lncRNA and mRNA expression profile and the impact on nucleus pulposus cell function after mir-155 overexpression lentivirus transfection.To verify the role and mechanism of GAS5 lncRNA in the nucleus pulposus cell apoptosis regulation,the apoptosis ratio of nucleus pulposus cells after GAS5 lncRNA overexpression was measured by Flow cytometry,and the expression of two key apoptosis related proteins,Caspase-3 and Bcl-2,were analyzed by Western Blot.[Results]A total of 631 up-regulated lncRNAs with unequivocal sequence information are found in the degenerated nucleus pulposus.There are 148 up-regulated lncRNAs contains178 binding sites of hsa-miR-155-5p,16 of which accord with Target Scan scoring standard,NR₀02819 contains six binding sites,both ENST00000511037 and NR₀27451 contains five binding sites,19 lncRNAs contains two binding sites,126 lncRNAs contains one binding site.Compared with negative control group,GAS5 lncRNA,with one hsa-miR-155-5p binding site,is down-regulated in the nucleus pulposus cells after exogenous transfection of hsa-miR-155 mimics?P<0.05?.LncRNA and mRNA microarray analysis reveals that there are 721 differentially expressed lncRNAs in the transfected nucleus pulposus cells?fold change no less than 2,P < 0.05?,of which 492 lncRNA is up-regulated,229 lncRNA is down-regulated.all of the 18 detected GAS5 lncRNA transcripts are down-regulated,among which just GAS5-021 alone has significant difference?fold change on less than 1.5,P<0.05?.Gene Ontology analysis showed that gene expression of nucleus pulposus cells is obviously changed after miR-155 overexpression,which focuses on the tumor,autoimmune diseases,degenerative diseases and cardiovascular diseases.Ankylosing spondylitis and synovitis seem to exert the most closely relation with the intervertebral disc.96 hours after GAS5 lncRNA overexpression lentivirus transfection,GAS5 lncRNA expression level is up-regulated in the nucleus pulposus cells?P<0.05?.The expression level of pro-apoptosis protein Caspase-3 is up-regulated,and anti-apoptosis protein Bcl-2 is just the opposite.Compared to negative control group,the apoptosis rate of nucleus pulposus cells is increased?P<0.05?.[Conclusions]LncRNA plays an important role in regulation of nucleus pulposus cells apoptosis during intervertebral disc degeneration,GAS5 lncRNA overexpression in the nucleus pulposus changes the expression of two key members of the family protein on mitochondrial apoptotic pathways.By up-regulation of Bcl-2 and down-regulation of Caspase-3,GAS5 lncRNA can activate the mitochondrial apoptotic pathways and promote apoptosis of nucleus pulposus cells.GAS5 lncRNA monitors the balance of pro-apoptosis and anti-apoptosis molecules on the mitochondrial apoptosis pathway;the status of GAS5 lncRNA expression determines the sensitivity of nucleus pulposus cells to apoptosis stimuli.Mi R-155 seems to participate in the apoptotic regulation of GAS5 lncRNA,while the mutual regulatory mechanism needs to be further studied.