Screening And Biological Function Study Of CircRNA In Adult Degenerative Scoliosis

Objective:High-throughput sequencing technique was used to analyze the circRNAs gene expression profile in peripheral blood of adult degenerative scoliosis?ADS?.The differentially expressed circRNAs was screened and the differentially expressed circRNAs was analyzed for biological functions.To construct a network map of circRNA-miRNA mutual regulation,to find differentially expressed circRNAs and miRNA binding sites,to explore the biological functions of differentially expressed circRNAs in ADS patients,and to find potential diagnostic markers with high sensitivity and specificity.To provide a theoretical basis for deeply understanding the mechanism,diagnosis and treatment of ADS at the level of transcriptomics.Methods:Peripheral blood?5 ml?was collected from patients with ADS,lumbar disc herniation?LDH?,and healthy controls,3 cases each group.Extracting the total RNA,and the concentration and purity of total RNA were detected by NanoDrop ND-1000 ultra-micro spectrophotometer.The integrity of total RNA was detected by denaturing agarose gel electrophoresis.Based on the results of total RNA quality analysis,the high-throughput sequencing technology was used to detect differential expression of circRNA by using the second-generation high-throughput Illumina HiSeq sequencing technology platform to detect the expression profile of circRNA.The differentially expressed circRNAs was analyzed by volcanic map,cluster,KEGG and GO biological enrichment analysis,miRanda and Target Finder software predict the binding sites of spliced circRNA to miRNA.IRES finder software seeks whether the circRNA contains the information of internal ribosome entry site?IRES?.Results:1.The total RNA content,concentration,purity and integrity of the three groups meet the technical requirements of high throughput sequencing.2.A total of 690 circRNAs were identified and 154 new circRNAs were discovered Among them,29 circRNAs root in intron splicing,653 circRNAs root in exon splicing,and 8 circRNAs root in intergenic_region were found,506 circRNAs may contain IRES,185 circRNAs may not contain IRES3.ADS group compared with the control group,690 circRNAs were detected,20 circRNAs were differentially expressed,of which 10 were up-expressed and 10 were down-expressed.LDH group compared with the control group,636 circRNAs were detected,15 circRNAs were differentially expressed,of which 7 were up-expressed and 8 were down-expressed.ADS group compared with the LDH group,636 circRNAs were detected,24 circRNAs were differentially expressed,of which 12 were up-expressed and 12 were down-expressed.4.CircRNA-miRNA was co-expressed network in the peripheral blood of ADS.A circRNA can bind to multiple miRNAs,and a single miRNA can bind to multiple circRNAs.The down-expressed hsa_circ₀006156 can bind to miR-30-3p,miR-15b-3p,miR-147a and miR-149-3p,and hsa_circ₀001900 can bind to miR-100-3p,miR-15a-3p and miR-21-3p.5.Enrichment analysis results:?1?GO enrichment analysis results are as follows:ADS group compared with the control group,A total of 738 biological processes of GO enrichment analysis,mainly white blood cell-cell adhesion,epidermal growth factor receptor signal Pathway,ERBB signaling pathway,methylation of CpG islands,lactic acid metabolism,glutathione biosynthesis,non-ribosomal peptide biosynthesis,regulation of histone H4 acetylation,negative regulation of histone acetylation,hyalurance Acid catabolism process,peptide biosynthesis process;135 cell components,mainly cytoplasmic vesicle membrane,secretory granule membrane,basal membrane network,granule vesicle shell,clathrin aptamer complex,cytoplasm,organelle 134 molecular functions,mainly protein binding,homologous protein binding,ligand-dependent nuclear receptor transcription coactivator activity,protein heterodimer activity,cytokine receptor activity,SMAD protein binding activity,cysteine Endopeptidase activity,enhancer binding.LDH group compared with the control group,A total 531 biological processes of GO enrichment analysis,mainly involved in the regulation of lysate assembly involved in necrotic apoptosis,processing of N-glycans,regulation of macrophage differentiation,regulation of cell necrosis,and ligation.Positive regulation of enzyme activity;92 cell components,mainly nuclear,nuclear membrane,organelle,centromere,ubiquitin ligase complex,ribonucleoprotein granule,chromosome;117 molecular functions,mainly purine nucleotides Binding,transcriptional coactivator activity,substrate-specific transporter activity,ATPase activity,cell adhesion molecule binding,nucleotide binding,RNA polymerase ? promoter proximal sequence-specific DNA binding,hydrolase activity,transcription Factor binding.ADS group compared with the LDH group,A total 1395 biological processes of GO enrichment analysis,including negative regulation of cell processes,negative regulation of biological processes,regulation of cell cycle,tissue morphogenesis,response of cells to stimulation,and protein metabolism.cell biosynthesis process,nucleic acid metabolism process;204 cell components,including macromolecular complexes,intracellular membrane-free organelles,cytoplasm,nucleus,cell membrane,chromosome,microtubule cytoskeleton,organelle,protein complex;There are 201 functions,including organic cyclic compound binding,nucleic acid binding,protein binding,and ligase activity.?2?The results of KEGG enrichment analysis were as follows:.ADS group compared with the control group,26 pathways were found in the KEGG enrichment analysis,including Epstein-Barr virus infection,HTLV-I infection,pyruvate metabolism,and phosphatidyl Alcohol signaling system,ECM-receptor interaction,glycerophospholipid metabolism,osteoclast differentiation,cell adhesion molecules?CAMs?,Rapl signaling pathway;LDH group compared with the control group,19 pathways were found in the KEGG enrichment analysis,mainly includes N-glycan biosynthesis,cytosolic DNA sensing pathway,RIG-I-like receptor signaling pathway,apoptosis,NF-?B signaling pathway,Toll-like receptor signaling pathway,TNF signaling pathway;ADS group compared with LDH,38 pathways were found in the KEGG enrichment analysis,including hematopoietic lineage,endocytosis,HTLV-I infection,pyruvate metabolism,Notch signaling pathway,RNA degradation,TGF-? signaling pathway,phosphatidylinositol Signaling system,glycerophospholipid metabolism,HIF-1 signaling pathway,FoxO signaling pathway,Wnt signaling pathway,Hippo signaling pathway,Ras signaling pathway,PI3K-Akt signaling pathway.Conclusions:1.In this study,the expression of circRNA in ADS peripheral blood was constructed for the first time based on the second generation high-throughput Illumina HiSeq sequencing technology.154 new circRNAs were found,which provided a theoretical basis for further research on the biological function of circRNAs.2.Multiple circRNAs are differentially expressed in ADS peripheral blood.Some circRNAs may contain IRES,which initiate protein translation.3.CircRNAs participate in the development progression of ADS by co-expression with miRNAs,and are expected to be biomarkers and target for early screening,diagnosis and treatment of ADS,and further research is needed.