Study On The Role Of MiR-198 In Intervertebral Disc Degeneration And Its Epigenetic Regulation

Intervertebral disc degeneration(IDD)belongs to the pathologic basis of spinal degenerative disease.Although many studies have reported the pathogenesis of IDD,but no one has clarified the detailed pathological mechanism.It is concluded that the IDD induced by complex factors,which changes in Extracellular matrix(ECM)for IDD pathological changes including a representative lesion.Related research showed that at the time of the occurrence and development of IDD,which contained in nucleus pulposus of type II collagen levels decrease gradually,type I collagen increased gradually.The two substances change degree and IDD development is positively related.Integrin is derived on the collagen receptor.Its in-nucleus pulposus cells are renowned for their integration through contains I / II type collagen and two subunits(alpha 2 beta 1 and alpha 1 beta 1)connected together,mediated by intracellular and extracellular information exchange,further to biological function.In this study,through the distribution and level of in-depth comparison in healthy intervertebral disc,degenerative intervertebral disc tissue mir-198,ITGA1,COL1A1 mRNA and protein coding were analyzed.Mir-198 was able to effectively regulation body and nucleus pulposus cell ECM integrin,type I collagen using the apparent mir-198 on genetic mechanism of the upstream regulatory role.If these questions were clarified,it will help in a related field,to further understand the exact mechanism of IDD disease occurrence and development of ECM changes.Furthermore,it will be helpful for mir-198 gene may become a clinical treatment targets for IDD lesions to give a scientific basis.The study consists of the following 4 experiments:Study on the expression of miR-198 and COL1A1 and ITGA1 in human nucleus pulposus Objective:To detect the expression level of miR-198 gene in the disease IDD significantly by the results of the previous microRNA chip detection technology.To explore target genes of mir-198 and potential mechanism of IDD Methods:Respectively obtain 5 cases IDD patients,5 cases of healthy intervertebral disc death patient specimens.Two groups of expression mir-198 gene,ITGA1 and COL1A1 mRNA levels was determined by quantitative real-time PCR.By applying Sirius red staining technique,the distribution of the two groups of collagen were investigated for the pathological changes of the two groups.By immunofluorescence staining technology,westen blot techniques the expression of integrin alpha 1,type I collagen level were detected in two groups.By immunofluorescent labeling techniques,the level of CO expression were detected in two groups targeting at mir-198 COL1A1 gene,ITGA1.Results:IDD type I collagen gradually began to take place of type II collagen and mir-198 reduced levels.ITGA1 and COL1A1 mRNA and corresponding protein expression level of ascent,fluorescence results indicate mir-198 gene showed positive cells of COL1A1 and ITGA1 the expression was negative and vice versa.Conclusion:The expression of miR-198 gene,ITGA1 and mRNA COL1A1 showed negative regulation within its corresponding protein miR-198 levels in the nucleus pulposus tissues.In vitro study on the regulation of COL1A1 and ITGA1 expression by experiment two miR-198 Objective:The miR-198 gene expression level was significantly reduced in IDD lesions.COL1A1 and ITGA1,two types of mRNA,correspondly increase in protein expression.But there are still the following questions,the result is due to the COL1A1 gene ITGA1,mRNA miR-198 as the role of the induced,or because of other intermediately linked cause? Using the bioinformatics technology,the existence of mRNA ITGA1,COL1A1 in the 3 ’UTR and miR-198 target genes were analyzed.Therefore,the second part by in vitro transfection control,dual luciferase reporter gene technology research mir-198 direct regulation ITGA1,COL1A1 mRNA,and analysis whether it will affect cell function characteristics.Methods:COL1A1,mir-198,mRNA ITGA1 3 ’UTR inner relationship were measured with dual luciferase reporter gene technology.In order to observe mir-198 in nucleus pulposus cells level change,Western blot to detect the mRNA expression of COL1A1 and ITGA1.Choosing real-time quantitative PCR test certificate of osteosarcoma cell line u-2os,MG-63 was expressed in the mir-198 gene.By applying Lentivirus on u-2os,MG-63 were transfected and to observe the changes of cell mir-198 level.Transwell test was applied to determined whether it will affect cell function characteristics.Results:MiR-198 was able to directly affect the COL1A1 and mRNA ITGA1 UTR 3 ’.The level of miR-198 gene was down regulated or up-regulated,which could regulate the expression of ITGA1 and COL1A1 in nucleus pulposus cells.The results show that compared to the miR-198 down group,miR-198 up regulation group U-2OS,MG-63 showed faster healing.In addition,miR-198 up regulation group U-2OS,MG-63 showed enhanced mobility,invasion rate.MiR-198 level down group MG-63,U-2OS showed a decline in the rate of migration,invasion rate.Conclusion:The miR-198 gene is directly affected by COL1A1,mRNA ITGA1 3 ’UTR,which can regulate the expression level of Integrin alpha 1 and Collagen I protein,and ultimately affect the functional characteristics of the cell.In vivo study of the regulation of COL1A1 and ITGA1 expression by experiment three miR-198 Objective:We know that the miR-198 gene can directly act on COL1A1,mRNA ITGA1 3 ’UTR two’ target genes by experiment one or two,and the above phenomena are verified by in vitro studies.But because the structure of the cells in vitro is usually simple,can better control the culture conditions,there is no more influence factors,therefore,can not compare the living environment of intervertebral disc tissue.In part three,C57 animal IDD model was used to study the level of miR-198 gene,and to investigate whether the miR-198 gene would affect the course of IDD disease.Methods:Real-time quantitative PCR technique was used to detect the expression of C57 mice mir-198 level,by the rat tail puncture build IDD models so that the transfection adeno-associated virus,reduced or increased intradiscal expression mir-198 level,by Western blot after transfection was measured 3,6,the 12 th node expression of integrin alpha 1,collagen I levels,combined with intervertebral height index,double labeling method,the organization score method understand the differences in different IDD node mice.Results:3 weeks,6 weeks and 12 weeks after modeling,the expression of integrin alpha 1,collagen I protein levels increased.Up regulation of /miR-198 gene can decrease the different time nodes in the expression of integrin alpha 1,collagen I levels.Compared to controls and mir-198 downregulated group,mir-198 upregulation of each node has a higher score,intervertebral height index.Conclusion:In vivo miR-198 gene can effectively protect the IDD,miR-198 down can exacerbate the evolution of IDD disease.Study on the mechanism of epigenetic regulation of miR-198 in Experiment four Objective:Through experiment two,the regulation mechanism of miR-198 gene in IDD nucleus pulposus was confirmed by experiment three.But it has not been clarified the mechanism of action,and the experiment four will study the regulation mechanism of miR-198 gene.Methods:By methyl of DNA MeDIP-PCR technology determining the adjacent sub regions of the four CpG island mir-198 two precursor promoter,culture 5Aza-CdR on the in vitro primary marrow nuclear cells was detected.QRT PCR determination was applied to understand the regulatory mir-198,COL1A1,ITGA1 mRNA situation.Results:The IDD group mir-198 two precursor start the would decline in COL1A1,ITGA1 mRNA level within 10 KB.Four possible methylation sites are more prone to methylation phenomenon,but the results are statistically different(P < 0.05)below only two sites:,the increase in the level of mir-198 expression of nucleus pulposus cells 5Aza-CdR.Conclusion:The abnormal expression level of miR-198 in IDD is closely related to the degree of methylation of the 2 CpG islands.