Circular RNA Profile And Functional Study Of Memory CD4+T Cells In Ankylosing Spondylitis

Ankylosing spondylitis(AS)is a common autoimmune disease,characterized by inflammation of sacroiliac joint and central axis spinal joint,and can lead to joint dysfunction and even loss of function,which seriously affects the normal work and life of the patients.The pathogenesis of AS is unclear,with 95 percent of the patients HLA-B27 positive.However,genome-wide association studies(GWAS)have shown that more genetic factors are involved in the pathogenesis of AS.Most sequences in the human genome cannot encode proteins,and these non-coding sequences also play an important role in the various life activities.Circular RNA(circRNA),as a non-coding RNA with special circular structure,is stable,conserved,and tissue development specific.circRNA plays an important role in the occurrence and development of many diseases in human beings and is a potential diagnostic biomarker and therapeutic target,but there is still a lack of relevant research on circRNA expression profile in AS.T cells are closely related to the pathogenesis of AS by secreting a variety of inflammatory factors such as TNF-α,IL-17,IL-23 or improving self-responsiveness to promote disease progression.Recent studies have focused on IL-17-producing Th17 cells.Some studies have shown that Th17 cells exhibit a memory phenotype and may reside in the memory T cell pool,whereas there are few related studies on memory CD4+T cells in AS.Therefore,this study established circRNA expression profils of memory CD4+T cells in AS to verify the differential expression of circRNA and further explore the function of circRNA in cells and whether it is involved in the pathogenesis of AS.Chapter I CircRNA expression profile and bioinformatics analysis in memory CD4+T cells of ankylosing spondylitisObjective:To establish circRNA expression profile of memory CD4+T cells in AS,and to predict the function of differentially expressed circRNA-related mRNA in cells,which will lay the foundation for further exploring the role and mechanism of circRNA.Methods:The peripheral blood of 5 AS patients without treatment and 5 healthy controls were collected,and the density gradient centrifugation method was used to obtain peripheral blood mononuclear cells(PBMC).The memory CD4+T cells were sorted The circRNA expression profile was established by high-throughput sequencing,and the differentially expressed circRNAs were screened.The sequencing results were further verified in the validation cohort.Biological information of differentially expressed circR-NAs predicts their potentially targeted miRNAs and mRNAs.KEGG and GO enrichment analyses of mRNAs to speculate on the possible biological functions of differentially ex-pressed circRNAs.Meanwhile,the receiver operation characteristics(ROC)curve was em-ployed to analyze whether the differential circRNAs had diagnostic value.Results:The expression of 10,808 circRNAs were detected in 10 samples,of which 357 were up-regulated and 278 were down-regulated.Further PCR showed that circPDE5A,circCCNY and circRASA1 were significantly elevated in AS.Bioinformatics prediction found that circPDE5A,circRASA1 and circCCNY might target 256 miRNAs and 6989 mRNA.The results of KEGG and GO analyses of relevant mRNA showed that differentially expressed circRNAs may be involved in biological processes such as cell metabolism,gene expression regulation through signaling pathways such as Akt,MAPK,Wnt,and autophagy.The areas under receiver operating characteristic curve(ROC)of circPDE5A,circRASA1 and circCCNY were 0.7850,0.7652 and 0.7565 respectively and statistically significant.Conclusion:The circRNA profile of memory CD4+T cells in AS patients was different from that in the healthy control group.The differentially expressed circRNAs were potential markers of AS diagnosis,and they may play a regulatory role in the pathogenesis of AS via multiple signaling pathways.Chapter Two:Construction and functional study of circRNA-miRNA-mRNA pathwayObjective:To construct circRNA-miRNA-mRNA pathway and explore its function in cells.Methods:The sequence information of circRASA1 and circCCNY were obtained ac-cording to circBase,and the sequence specificity was verified by Sanger sequencing of PCR products.RNase R treated RNA samples for PCR reaction and agarose gel electrophoresis to detect the stability of circRNA.Dual luciferase reporter assay verified the combination of circRASA1 and circCCNY with miR-181a/b/c/d-5p.Jurkat cells were transfected with len-tivirus to establish circRASA1 and circCCNY low expression stable cell lines,and the ex-pression levels of miR181c-5p and PKC-8 were detected by real-time PCR and Western blot.The expression level of PKC-δ was analyzed after overexpression of miR-181c-5p by trans-fecting miR-181c-5p mimic.KEGG analyzed the relevant pathways of PKC-δ.In the above models,the expression of autophagy-related genes was detected by real-time PCR and west-ern blot.Results:Sanger sequencing verified that the PCR amplification fragment of circRASA1 and circCCNY contained back-splicing sites.The internal reference molecule GAPDH was significantly degraded after RNase R treated the samples,whereas circRASA1 and circCCNY were relatively stable.Dual luciferase reporter assay showed that circRASAl,circCCNY and miR-181c-5p had stronger binding force,and the expression level of miR-181c-5p in memory CD4+T cells of AS patients was significantly reduced,Interfering with the expression of circRASA1 and circCCNY could upregulate the expression of miR-181c-5p,while downregulating the expression of PKC-δ mRNA and protein.Overexpression of miR-181c-5p could also downregulate the expression of PKC-δ protein.KEGG analysis showed that PKC-δ was involved in autophagy-related pathways.Interfering with circRASA1 and circCCNY expression could downregulate the mRNA and protein levels of autophagy-related genes ATG5 and ATG7 and protein expression of LC3B-Ⅱ.Overexpres-sion of miR-181c-5p could similarly downregulate the protein expression of ATG5,ATG7 and LC3B-Ⅱ.Conclusion:circRASA1、circCCNY can competitively bind to PKC-δ miR-181c-5p,regulate the expression of autophagy-related genes,altering autophagy level of cell and pos-sibly affecting cell autoreactivity and thus participating in AS pathogenesis.