The Study On The Regulation Of Intervertebral Disc Degeneration By Silencing NDC80,RAD21 And BUB1B Genes

【Objective】Back pain has become one of the most common musculoskeletal disorders in the world,and symptomatic intervertebral degenerative changes are considered to be the most important and important factor.This study based on the bioinformatics analysis of gene expression of intervertebral disc degeneration in the Gene Expression Omnibus.Our group screened the differentially expressed miRNA targeted genes,found the upregulation of NDC80,BUB1 B and RAD21 genes.We designed RNAi oligonucleotide sequence corresponding to the above three genes respectively,and the effective silence RNAi fragments were screened in the nucleus pulposus.Then we investigated that effect of NDC80,BUB1 B and RAD21 Gene silence on nucleus pulposus cell cycle distribution,apoptosis,and the expression change of type II collagen.The improved laminectomy was used to influence post-spinal column stability to construct an intervertebral disc degeneration rat model,and the expression levels of NDC80,BUB1 B and RAD21 in intervertebral disc nucleus were observed.The purpose of this study is to explore the pathophysiological and molecular mechanisms of intervertebral disc degeneration in order to develop genetic diagnosis and new approaches on gene therapy.【Method】1.Bioinformatics analysis: Gene expression data set GSE34095,GSE63492 and GSE45856 were downloaded from Gene Expression Omnibus.Raw data were converted into recognizable format with package affy of R,and missing values were then imputed.After data normalization with median method,differential analysis was performed using limma package for degeneration samples and control,respectively.|Log（fold change）| > 1 and adjust P value < 0.05 were set as the cut-offs to screen out DEGs and differentially expressed miRNAs.Target genes of differentially expressed miRNAs were screened by miTarBase and Targetscan.In order to identify disturbed biological functions in IDD,GO functional enrichment analysis was performed for DEGs using DAVID with a threshold of P < 0.05.In order to correctly uncover and annotate all functional interactions among proteins in the human IDD,we applied the STRING online tool to analyze the PPI of DEGs with experimentally validated interactions.2.Experimental study in vitro: According to the mRNA sequences of NDC80,BUB1 B and RAD21 genes（NM₀06101,NM₀01211 and NM₀06265）in Genbank,the siRNA of NDC80,BUB1 B and RAD21 genes（each siRNA had three pairs of sequences）and negative controls（NC）were designed and synthesized it.The 3rd generation of NP cells was used for transfection.Cells were assigned into blank group（cells with no transfection）,NC group（cells transfected with empty vector）,siRNA-NDC80 group（cells transfected with siRNA-NDC80-1,siRNA-NDC80-2 and siRNA-NDC80-3 respectively）,siRNA-BUB1 B group（cells transfected with siRNA-BUB1B-1,siRNA-BUB1B-2 and siRNA-BUB1B-3 respectively）and siRNA-RAD2 group（cells transfected with siRNA-RAD21-1,siRNA-RAD21-2 and siRNA-RAD21-3 respectively）.The siRNA with highest transfection efficiency was selected by quantitative real-time polymerase chain reaction（qRT-PCR）as targeted plasmid.After NDC80,BUB1 B and RAD21 gene silencing,MTT assay was used to test the proliferation of cells,flow cytometry was used to detect cell apoptosis to detect the effect on cell cycle distribution and apoptosis and the the content of collagen II.3.Experimental study in vivo: Animal models of intervertebral disc degeneration were constructed through removing the 1-6 spinous process,vertebral plate,ligamentum flavum and part of the zygapophyseal joint.The animal model was constructed after 3 months.Morphological differences between normal intervertebral discs and degenerated intervertebral discs were observed by HE staining.qRT-PCR and immunohistochemistry were used to detect the differences in the expression of NDC80,BUB1 B and RAD21 in the normal group.【Results】1.Bioinformatics analysis: For GSE34095,a total of 153 DEGs were screened out from NP cells expression profile,which including 111 up-regulated genes and 42down-regulated genes.Among them,RAD21,NDC80 and BUB1 B genes were upregulated.For GSE63492,7 up-regulated miRNAs and 8 down-regulated miRNAs were obtained from degenerative NP cells samples compared with the controls,and miR-5100 was one of the down-regulated miRNAs.Furthermore,for GSE45856,a total of 6 up-regulated miRNAs and 9 down-regulated miRNAs were obtained from degenerative NP cells samples compared with the controls.Among them,miR-1246 and miR-3908 were down-regulated miRNAs.Based on prediction analysis,the targeted genes of miR-5100,miR-1246 and miR-3908 were RAD21,NDC80 and BUB1 B respectively.Therefore,RAD21,NDC80 and BUB1 B genes expressed highly in other profiling data.GO enrichment analysis found that RAD21 and BUB1 B genes were involved in cell cycle,mitotic cell cycle,cell apoptosis,programmed cell death and cell death;and NDC80 gene was participated in cell cycle and mitotic cell cycle.KEGG pathway enrichment analysis revealed that RAD21 and BUB1 B genes were involved in cell cycle related signaling pathways.The PPI networks of DEGs from NP cells and AF cells noted that the NDC80,BUB1 B,and RAD21 were the hub genes in the PPI network of NP cells.2.Experimental study in vitro: The primary NP cells grew slowly;after cell adherence,it was observed to stretch out the pseudopodia and transform into multangular or oval cells,then cells were extended and arranged in long spindle shape.The 3rd generation of NP cells grew rapidly;after cell adherence,the pseudopodia was stretched out,and cells were extended and showed spindle or multangular shaped;cells were in typical cobblestone appearance.After transfection for 48 h,there was no difference of gene expression between the blank and NC groups in three genes（NDC80,BUB1 B and RAD21）（all P < 0.05）.Compared to the blank and NC groups,the mRNA expression decreased in all siRNA groups（all P < 0.05）,and the most obvious changes were found in the siRNA-NDC80-1,siRNA-BUB1B-2 and siRNA-RAD21-2 groups,which indicated the highest transfection efficiency.Compared to the blank and NC groups,cells of the siRNA-NDC80,siRNA-BUB1 B and siRNA-RAD21 groups was decreased in G1 phase and increased in S phase（P <0.05）,while no obvious changes was observed in M phase（P > 0.05）.The apoptosis rates also decreased（P < 0.05）.Results reflected that NDC80,BUB1 B and RAD21 gene silencing accelerates cell cycle and inhibits cell apoptosis and increased the content of collagen II.3.Experimental study in vivo: The clear structure of intervertebral disc,regular arrangement of annulus fibrosis,evenly distributed NP cells and regular cartilage endplates with concentric circle pattern were observed in the normal group.While in the IDD group,the pathological changes of the intervertebral disc including: reduced plate height,the fiber ring was obviously thickened and irregular,and the fibers disintegrate,shrink of NP,decreased number and uneven distribution of NP cells,unclear boundary of NP and annulus fibrosis,derangement of the collagen of the annulus fibrosis,calcification,ossification and separation of the cartilage endplate.And the expressions of NDC80,BUB1 B and RAD21 are higher in IDD group than normal group.【Conclusion】1.According to the results of bioinformatics analysis,this study puts forward the scientific hypothesis: NDC80,BUB1 B and RAD21 gene play a potential regulatoryrole in the process of intervertebral disc degeneration.Through silence NDC80,BUB1 B and RAD21 gene of nucleus pulposus cells,we found that gene silencing accelerates cell cycle and inhibits cell apoptosis and increased the content of collagen II.This preliminary confirmed the regulatory function of these genes on nucleus pulposus cells.In vivo study,the expression levels of NDC80,BUB1 B and RAD21 in the degenerative disc tissues were significantly up-regulated,which further confirmed the scientific hypothesis that it regulates the progression of intervertebral disc degeneration by regulating the phenotype of the nucleus pulpocyte.The study of the pathophysiological and molecular mechanisms disclosed by the institute provides a theoretical basis for the development of the disease genetic diagnosis and the gene therapy.2.In this study,the improved laminectomy was used to influence post-spinal column stability to construct an intervertebral disc degeneration rat model,and the effectiveness of the model was evaluated by HE staining,immunohistochemical staining and qRT-PCT.This method is simple,scientific,effective and provide the ideal animal model for the same researching field,in order to study the pathophysiology and molecular mechanism of intervertebral disc degeneration.