Preliminary Screening And Molecular Network Function Analysis Of Peripheral Blood Lymphocyte CircRNA Expression In Patients With Spinal Tuberculosis

Part ? Screening and validation of lymphocyte-specific circRNAs from spinal tuberculosisBackground: Spinal tuberculosis?STB?is a common form of extrapulmonary tuberculosis?TB?,and China is one of the countries with the highest incidence of tuberculosis,second only to India.The most common manifestation of spinal tuberculosis is chronic low back pain,because the diagnosis of spinal tuberculosis is easily overlooked,and the results often lead to the loss of neurological function and the development of spinal bone deformity.Due to the gradual increase in multidrug-resistant tuberculosis strains,the risk of treatment for tuberculosis is increasing.At present,for spinal tuberculosis,the first line of treatment is still anti-tuberculosis drug treatment,and the surgical intervention is applicable to the decompression of nerve elements that do not respond to drug treatment,the recovery of spinal stability and the correction of deformity.Spinal tuberculosis seriously affects the dailylife of patients and increases the economic burden of society.Therefore,studying its detailed pathogenesis is of great significance for the early diagnosis and treatment of diseases.However,the genetic pathogenesis of spinal tuberculosis is still unclear.At present,with the development of bioinformatics,the impact of non-coding RNA on spinal diseases is gradually manifested,and the genes are mutually regulated,interconnected,and mutually restricted and regulated to participate in the occurrence of spinal diseases.development of.Small RNA has been reported to play an important role in the pathogenesis of tuberculosis,but it has not been reported in the case of spinal tuberculosis.In recent years,with the rapid development of genomics,humans have continuously discovered that small RNA plays a key role in the pathogenesis of various diseases,such as brain,lung cancer,colon cancer,and degenerative knee arthritis.Functionally,miRNAs direct the silencing complex?RISC?to degrade mRNA or hinder translation by base pairing with the target gene mRNA.Recent studies have shown that circRNA is mainly distributed in the cytoplasm,the series is highly conserved,abundant and stable in mammals,with certain tissue,timing and disease specificity,and the circRNA molecule is rich in microRNA?miRNA?binding sites.The cell acts as a miRNA sponge,which in turn releases the inhibitory effect of the miRNA on its target gene and increases the expression level of the target gene.This mechanism of action is called a competitive endogenous RNA?ceRNA?mechanism.CircRNA plays an important regulatory role in disease through miRNA interactions associated with disease.To this end,we try to find key circRNA from the peripheral blood of patients,and to find a network of circRNA-miRNA-mRNA regulation,which can effectively help the pathogenesis,early diagnosis and prognosis of the disease.Therefore,studyingthe above circular RNA will help us to understand the pathogenesis of spinal tuberculosis.Objective: Spinal tuberculosis is a common and frequently-occurring disease in orthopedics.Early diagnosis and treatment are important for the prognosis of spinal tuberculosis.At present,spinal tuberculosis mainly depends on clinical manifestations,laboratory tests and imaging examinations.However,in the early stages of the disease,typical clinical symptoms,laboratory tests,and imaging lack specificity,so it has an impact on its diagnosis and treatment.Therefore,our aim was to investigate the screening of differentially expressed circular RNAs and to explore specific circulating circRNA markers that have potential diagnostic value for spinal tuberculosis.Methods: This study used high-throughput analysis of human circRNA from Arraystar,USA.This study used a two-stage case-control study design.The first stage is the initial screening stage.Three pairs of samples of new spinal tuberculosis lymphocytes and healthy adult lymphocytes are collected.The circRNA microarray is used to detect the circRNA expression profile in the cells,and the differentially expressed circRNAs are screened.The screening standard is the fold difference of expression?fold?Change,FC)> 1.2 times,P <0.05.The second stage is the independent verification stage,which collects 25 pairs of new spine tuberculosis lymphocytes and healthy adult lymphocyte samples,and uses quantitative reverse transcription polymerase chain reaction?RT-PCR?technique to screen the initial screening.Candidate circRNAs screened at the stage were validated for validation studies.Results: 89 circRNAs with differential expression were screened by circRNA microarray,of which 46 were up-regulated and 43 were down-regulated.The RT-PCR results of the selected 4 circRNAs with thehighest difference in differential expression between the up-regulated and down-regulated spinal tuberculosis patients showed that hsa_circRNA₁00632expression was up-regulated in spinal tuberculosis patients compared with healthy adults.The expression of hsa_circRNA₄00770 and hsa_circRNA₄03882 was down-regulated,which was consistent with the chip detection results.A molecular regulatory network with circRNA as the core was constructed,and the regulatory relationship between circRNA-miRNAs in spinal tuberculosis was presented.Conclusions: 1.In this experiment,the expression profile of circular RNA in peripheral blood lymphocytes of spinal tuberculosis was identified.There were 89 circRNA expression differences,of which 46 circular RNAs were up-regulated and 43 circular RNAs were down-regulated.Real-time quantitative PCR was used to verify the expression of hsa_circRNA₁00632 in the spinal tuberculosis group,and hsa_circRNA₄00770 and hsa_circRNA₄03882 were decreased in the spinal tuberculosis group.Part ? Functional prediction analysis and molecular regulation network analysis of breast cancer-related circRNAsBackground: CircRNA has a "miRNA cavernous" effect or can act as a competitive endogenous RNA?ceRNA?to inhibit its activity by binding to miRNA,thereby regulating the expression of miRNA target genes and affecting the normal physiological functions of the human body.It is related to the occurrence and development of various diseases.This study used bioinformatics analysis strategies to predict the function of differentially expressed circRNAs and explore their mechanisms of action in the pathogenesis of spinal tuberculosis.Objective: To construct a circRNA-miRNA regulatory network with circRNA as the core,and to explore its pathogenesis by using bioinformatics methods,and to provide ideas for similar studies of other diseases.Methods: The differential expression profiles of spine tuberculosis lymphocytes circRNAs screened in the first part using circRNAs were performed on the gene ontology of differential circRNA-related genes using the Database for Annotation?Visualization and Integrated Discovery,DAVID?.Ontology,GO)and the Kyoto Encyclopedia of Gene and Genomes?KEGG?analysis predict the relevant functions and signaling pathways of target genes.Using TargetScan,miRanda's self-made miRNA target prediction software predicts circRNA-microRNA interactions and constructs a circRNA-miRNA regulatory network.Results: GO analysis showed that the most enriched biochemical process?BP?in circRNAs differentially expressed by spinal tuberculosis lymphocytes is mainly involved in cellular response to DNA damage stimulus,peptidylserine phosphate?peptidyl-serine hosphorylation?,DNA repair,?proteinubiquitination?,positive regulation of NF-kappaB transcription factor activity,DNA damage response,Signal transduction by p53 class mediator resulted in cell cycle arrest,protein phosphorylation.Cellular components?CC?mainly involve chromatin,nucleoplasm,cytoplasm,centrosome,membrane,cleavage furrow,focal adhesion.Lamellipodium,Cul3-RING ubiquitin ligase complex,cytosol.Molecular function?BF?ubiquitin-protein transferase activity,kinase activity,ATP binding,chromatin binding,protein binding.The results of KEGG pathway analysis showed that Epstein-Barr virus infection,Pancreatic cancer,and Sphingolipid signaling pathway were associated with differential circRNAs.A single circRNA has a matching binding site with multiple miRNAs.Conclusions: 1.CircRNAs participate in spinal tuberculosis-related signaling pathways,which can be combined with multiple spinal tuberculosis-related miRNAs to participate in the development and progression of spinal tuberculosis.2.In this experiment,differentially expressed circRNA-related target genes were analyzed by KEEG pathway,and three possible regulation pathways of spinal tuberculosis were obtained.The above three regulatory pathways may be used to promote the development of pathological mechanisms of spinal tuberculosis.effect.