The Study On Mechanism Of MiR-623 In Intervertebral Disc Degeneration By Targeting IGFBP5

Objective: Intervertebral disc degeneration(IDD)is the pathological basis of various spinal degeneration-related diseases,and the pathogenesis related to degeneration has not yet been elucidated.Among the factors affecting the degeneration of the interstitial disc,the most important factor is the degeneration of the nucleus pulposus cells,which leads to changes in related pathological mechanisms.regulation,resulting in changes in related signaling pathways.miRNAs regulate the growth and development of cells or tissues at specific stages by targeting m RNA binding sites and affecting their transcriptional processes.In this study,the IDD-related miRNAs in nucleus pulposus cells were predicted by bioinformatics,miRNA transfection was used and luciferase was used for gene targeting verification,and q PCR,western-blot and other related technologies were used to find and verify related micro RNAs Targeting changes in the chemical composition of nucleus pulposus cells.Through human intervertebral disc experiments,in vitro cell experiments,and animal experiments,the mechanism of action of miRNA in intervertebral disc degeneration has been further confirmed.At the same time,it provides a new gene drug treatment plan for the prevention,diagnosis and treatment of intervertebral disc degeneration.Research methods: The first part,bioinformatics analysis and verification,preprocessed the data in the three datasets GSE63492,GSE19943 and GSE116726 downloaded from the GEO database,and then used the limma package to analyze the differentially expressed miRNAs of the IDD and Helaty groups.Then,three sets of differential miRNA intersections were taken,and predicted genes were predicted from Targetscan,Mirtar and miRDB databases.Differential expression analysis was performed using GSE23130 with the aim of validating the predicted gene results.The key miRNAs were verified,and the intervertebral disc tissues of IDD patients and patients without intervertebral disc degeneration were taken,and q RT-PCR was used to detect whether there were differences in the expression of key miRNAs.The second part of cell experiments,in order to further understand the mechanism of miRNA action,we verified by cell experiments: human nucleus pulposus cells(HUM-iCell-s012)were added with TNF-α at a concentration of 20ug/L and treated for 24 h to construct intervertebral disc nucleus pulposus.Cell degeneration model;the expression of apoptosis-related proteins caspase3 and bax in the experimental group and the control group were determined by western-blot technology;mimics NC,hsa-miR-623 mimics,inhibitor NC,hsa-miR-623 inhibitor Normal nucleus pulposus cells and nucleus pulposus cells treated with tumor necrosis factor for 24 h were respectively transfected;48h after transfection,the expression levels of collagen Ⅱ,aggrecan,IGFBP5 and other related results were detected by western blot.Normal nucleus pulposus cells were used to verify the targeting relationship between miR-623 and IGFBP5 by dual-luciferase reporter gene assay;The expression of IGFBP5 at gene and protein level was further verified by q RT-PCR,and the expression levels of collagen Ⅱ and aggrecan were also checked.In the third part of the animal experiment,a rat model of intervertebral disc degeneration was constructed by acupuncture,and the expressions of apoptosis-related proteins caspase3 and Bax in the experimental group and the control group were measured by western-blot technology,respectively,and hsa-miR-623 mimics,hsa-miR-623 inhibitor,the expression levels of collagen Ⅱ and aggrecan were detected by q RT-PCR and western blot.Result:In the first part,hsa-miR-623 and hsa-miR-508-5p were obtained as key miRNAs through GEO database,and 8 genes co-regulated by key miRNAs were predicted in Targetscan,Mirtar and miRDB databases as predicted genes.IGFBP5 was identified as the target gene for this study.miR-623 expression is elevated in degenerated disc tissue.In the second part,the NCBI website predicted that IGFBP5 has a binding site with miR-623,and the expression of IGFBP5,m RNA and protein levels decreased in the nucleus pulposus cells of IDD patients,showing an inverse correlation with miR-623.The expression of caspase3 and Bax in the nucleus pulposus cells of the degeneration group increased;in the luciferase experimental study,it was found that compared with the negative control group of mimics,the luciferase activity of miR-623 mimics on the wild IGFBP5 3’-UTR was significantly reduced.The fluorescence activity of the mutant group did not change ;the nucleus pulposus cells were transfectedwith mimics NC,hsa-miR-623 mimics,inhibitor NC,and hsa-miR-623 inhibitor.miR-623 mimics reduced the expression of collagen II,aggrecan and IGFBP5.Then decreased the activity of nucleus pulposus cells;transfection of hsa-miR-623 inhibitor resulted in the increased expression of collagen II,aggrecan and IGFBP5.In the third part,after the successful modeling of the rats,the expression of caspase3 and Bax in the degeneration group was checked to increase;the rats transfected with hsa-miR-623 inhibitor in the degeneration group were compared with the blank control,and the results showed that aggrecan,the expression level of collagen Ⅱ was up-regulated,and the change was more obvious compared with the normal group.Conclusion: miR-623 is highly expressed in degenerated nucleus pulposus;miR-623 affects nucleus pulposus cell degeneration by targeting IGFBP5.