| Background:Systemic lupus erthyematosus(SLE)is an autoimmune disease in which the immune system attacks healthy cells and tissues throughout the body.Currenntly,many scholars generally believe that the occurrence of SLE is influenced by multiple complex factors such as heredity,environment and immunity.The patientis of SLE is easy to relapse,and the clinical treatment is still dominated by hormones,immunosuppressants,anti-malarial drugs,non-steroidal anti-inflammatory drugs and other methods,but there is still no radical cure.However,the side effects caused by long-term use of drugs can cause serious physical damage to patients and even organ failure.Therefore,it is of great significance to have a deeper understanding of the pathological mechanism of SLE and to find efficient therapeutic targets.A large number of studies have confirmed that circ RNAs(circ RNAs)play a crucial regulatory role in various human diseases,including autoimmune diseases.Hsa＿circ＿101053 is generated by circRACGAP1 exon cyclization,named circRACGAP1.It has been reported that circRACGAP1 is up-regulated in non-small cell lung cancer,gastric cancer and other tumors,and is involved in the regulation of physiological processes such as autophagy,apoptosis and chemotherapy resistance of tumor cells.However,there are few studies on the biological function and mechanism of circRACGAP1 in SLE.miR-22-3p is a novel autoimmune regulatory factor that plays an important role in lymphocyte biology and immune response.For example,Lin et al.found that miR-22 can inhibit the proliferation of fibroblast-like synovial cells and the differentiation of Th17 cells by inhibiting the transcription of Cyr61,and participate in joint inflammation and injury in rheumatoid arthritis.A previous study showed that miR-22 can promote monocyte differentiation by degrading MECOM,inhibiting the expression of GATA2 regulated by C-Jun.Phosphatase gene PTEN,located on human chromosome 10q23.3,is a major negative regulator of the upstream of PI3K/AKT signaling pathway,and is involved in various physiological processes such as cellular immune regulation,signaling,and immune response.By previous literature survey and bioinformatics analysis,the potential complementary sequences between circRACGAP1 or PTEN and miR-22-3p were discovered,suggesting that it may be form a ‘circ RNA-miRNA-m RNA’ regulatory network.However,is this mechanism present in SLE? What kind of physiological function does it have? We still need to clarify further.Methods:(1)The head-to-tail splicing junction sequence in the q RT-PCR product of circRACGAP1 was confirmed by Sanger sequencing.The peripheral blood mononuclear cell(PMBCs)isolated from diagnosed SLE patients(n=30)and healthy controls(n=28),as well as SLE patients before and after drug treatment(n=6).The expressions of circRACGAP1 and host gene RACGAP1 were examined by Q-PCR.The correlation between circRACGAP1 and SLE disease activity index(SLEDAI)or ds DNA or C3 were analyzed.Receiver operating characteristic(ROC)curve assesse the the clinical value of circRACGAP1.After UVB treatment,the level of circRACGAP1 was examined.Jurkat cells were transfected with circRACGAP1 overexpression vector,and the effects of circRACGAP1 on UVB-mediated apoptosis of Jurkat cells were detected by Flow cytometry,TUNEL and Western blot.(2)The subcellular localization of circRACGAP1 in Jurkat cells was detected by nuclear cytoplasmic isolation.Bioinformatics software was used to analyze the binding site between circRACGAP1 and miR-22-3p,and the expression regulation relationship between circRACGAP1 and miR-22-3p was further verified by double luciferase reporter gene and Q-PCR.Q-PCR detected the expression of miR-22-3p in PBMCs SLE patients and healthy controls,as well as SLE patients before and after drug treatment.Clinical correlation of miR-22-3p with SLEDAI score,ds DNA and C3 level were determined.ROC curve analysis of circRACGAP1 clinical value.Jurkat cells were transfected with miR-22-3p antagomir,and the effects of miR-22-3p on UVB-mediated apoptosis of Jurkat cells were detected by Flow cytometry,TUNEL and Western blot.(3)Bioinformatics software analyzed the binding site between miR-22-3p and PTEN,and double luciferase,Q-PCR and Western blot were used to verify the binding and regulatory relationship between of the two.Q-PCR detected the expression of PTEN in PBMCs SLE patients and healthy controls,as well as SLE patients before and after drug treatment.Clinical correlation of PTEN with SLEDAI score,ds DNA,C3 level and expressions of circRACGAP1 and miR-22-3p were determined.By intervening circRACGAP1/miR-22-3p alone or simultaneously intervening circRACGAP1 and miR-22-3p in PBMCs of SLE patients/healthy volunteers and and UVB-treated Jurkat cells,western blot was used to detect the proteins of PTEN/AKT pathway and apoptotic proteins.Results:(1)circRACGAP1 was significantly decreased in PBMCs of SLE patients,and was negatively correlated with SLEDAI score and anti-ds DNA,and positively correlated with C3 level.The expression level of circRACGAP1 was increased with remission of SLE.The area under the ROC curve was 0.709,and the 95% confidence interval was 0.58～0.78.UVB irradiation significantly reduced the expression of circRACGAP1 in Jurkat cells,and promoted the cell apoptosis and the protein levels of Bax and cleaved-caspase 3 and decreased the protein level of Bcl2 in Jurkat cells.However,transfection of circRACGAP1 overexpressed vector significantly reversed UVB irradiation mediated the promoting roles of cell apoptosis,protein levels of Bax and cleaved caspase 3 and the inhibitory role on Bcl2 level.(2)Circ RACGAP1 is mainly expressed in the cytoplasm.Bioinformatics analysis and dual luciferase reporter genes confirmed the interaction between circRACGAP1 and miR-22-3p.Compared with healthy volunteers,the expression of miR-22-3p was significantly increased in PBMCs of SLE patients,and positively correlated with SLEDAI score and ds DNA,while negatively correlated with C3 level.miR-22-3p was decreased significantly in SLE patients after remission.The area under the ROC curve was 0.698,and the 95% confidence interval was 0.62～0.83.Results of flow cytometry and TUNEL assays results showed that silencing miR-22-3p significantly reversed UVB-mediated Jurkat cell apoptosis,and diminished UVBmediated up-regulation of Bax and cleaved caspase 3 and downregulation of Bcl2(3)Bioinformatics and double luciferase reporter genes verified the binding relationship between miR-22-3p and PTEN.Q-PCR and Western blot analysis confirmed that overexpression of miR-22-3p inhibited the m RNA and protein levels of PTEN,while silencing of miR-22-3p had the opposite effects.The results of QPCR showed that PTEN expression in PBMCs was downregulated in SLE patients compated to healty controls,and it could be increased after remission of the disease.Correlation analysis showed that PTEN expression level was negatively correlated with SLEDAI score,ds DNA and miR-22-3p expression,and positively correlated with C3 level and circRACGAP1 expression.Western blot analysis showed that circRACGAP1 promoted the expression of PTEN and Bcl2 in PBMCs and UVBtreated Jurkat cells,inhibited AKT phosphorylation and protein levels of Bax and Cleaved caspase 3,whereas,these results were reversed by the co-overexpression of miR-22-3p.Conclusion:In summary,the results indicate that circRACGAP1 and PTEN were downregulated in PBMCs in SLE patients,while miR-22-3p was upregulated,suggesting that circRACGAP1,miR-22-3p and PTEN were involved in the occurrence of SLE disease.Mechanistic results showed that circRACGAP1 promoted the expression of PTEN by sponging of miR-22-3p,inhibiting the activation of AKT pathway,eventually alleviating UVB induced apoptosis of Jurkat cells.These results proposed a novel ‘circ RNA-miRNA-m RNA’ molecular regulatory network,which further lays a foundation for enriching the biological function of circ RNA in SLE and provides potential targets for biological diagnosis and treatment of SLE. |
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