| Systemic lupus erythematosus(SLE)is a chronic autoimmune disease that causes immune damage to multiple organs throughout the body.It is characterized by abnormal lymphocyte activation,the production of high titer autoantibodies,the activation of complement,and extensive deposition of immune complexes.The disease is more common in Asian populations,especially at high altitudes in China,where the quality of life is relatively poor and places a heavy burden on families and society.Although SLE has a relatively mature treatment system,the pathogenesis of SLE is still unclear at present.It is crucial to maintain B-cell homeostasis,control Bcell overactivation and reduce impaired tolerance.More clinical data and experiments are needed to provide guidance.Therefore,the occurrence and development of SLE is a very complex pathological process,and there may be other pathogenesis.miRNA is a kind of 19-24 base length non-coding conserved RNA,abundant in eukaryotes,which can directly inhibit the function of mRNA or directly promote the degradation of mRNA by regulating the 3 ’unencoded region(3’UTR)of the downstream target gene mRNA.miRNA can be involved in regulating the expression,transcription,translation and modification of nearly one-third of target genes in the body,and it plays an important biological role in the development of the body and the proliferation and apoptosis of cells.More and more evidence shows that the regulatory mechanism in which miRNA is involved is an important way to regulate gene expression level.During the pathogenesis of systemic lupus erythematosus,a variety of miRNAs are related to the activity of the disease.The role of miRNA in SLE may become a valuable biological marker to provide more help for the diagnosis and treatment of SLE.In addition,both miRNA-150-5p and its downstream STAT1 have been shown to be involved in the pathogenesis of SLE,but there has been no study on this regulatory axis in the pathogenesis of SLE at present.The purpose of this study was to clarify that the differentially expressed miRNAs in SLE and the regulation of STAT1 expression by miR-150-5p affect the activation of B cells in SLE.Objective:To screen miRNAs that are differentially expressed in systemic lupus erythematosus and can be used as biological markers.miRNAs differentially expressed in peripheral blood PBMC of SLE patients were screened.To predict and investigate the biological functions of miR-150-5p and STAT1;To verify the biological mechanism of miR-150-5p regulating STAT1 regulating B cell activation.Methods:miRNA,SLE and biomarker were screened by meta-analysis.Peripheral blood of 15 SLE patients and 15 healthy controls was used to verify several miRNAs with strong specificity and sensitivity in the meta-analysis results by PCR.Through bioinformatics analysis,SLE and miRNA were analyzed to screen out miRNA and downstream target genes in multiple databases,and enrichment analysis of GO and KEGG was performed.Subsequently,peripheral venous blood of 3 active SLE patients and 3 healthy controls was collected.miRNAs with significant differences in PBMC were screened by second-generation sequencing technology.miRNA and DEGs with significant differences were analyzed by GO and KEGG and compared with all previous results to obtain target miRNAs.Downstream target genes were predicted by TargetScan,RNA22,miRBase,PITA and other miRNA databases.In MRL/lpr mice,the effects of miR-150-5p on STAT1 regulation were verified by PCR,flow cytometry,Western blot,ELISA,rescue experiment and luciferase reporter gene assay.Results:A meta-analysis of 30 miRNAs from 11 conforming literatures found that 17 up-regulated miRNAs and 13 down-regulated miRNAs may be used as diagnostic biomarkers for SLE.The highest sensitivity of miR-20a and miR-16 was 0.98(95%CI 0.89-1.00).The sensitivity of miR-221-5p,miR-380-3p,miR-556-5p,miR-758-3p,and miR-3074-3p combinations was also 0.98(95%CI 0.87-1.00).The specificity of miR-16,miR-15b and miR-150 was also 0.95(95%CI 0.75-1).Through PCR,we detected 14 miRNAs in peripheral blood of 15 SLE patients and 15 healthy controls,and found significant differences in 8 miRNAs(miR-150-5p,miR-16,miR20a,miR-15b,miR-146a,miR-155,miR-23a and miR-223)(P<0.05).miR-233 was up-regulated,the rest of miRNAs were down-regulated,and miR-150-5p showed the most significant difference and the difference multiple was the largest.After PBMC transcriptomic sequencing,10 differentially expressed miRNAs were found,including 3 down-regulated miRNAs and 7 up-regulated miRNAs.miR-150-5p was one of the significantly down-regulated miRNAs.Enrichment analysis showed that the differentially expressed miRNAs were related to inflammation,cell surface antibodies and multiple immune-related pathways.It was predicted that STAT1 may be involved in the pathogenesis of SLE and is regulated by miR-150-5p.The expression level of miR-150-5p in MRL/lpr mice was significantly lower than that in the control group(P<0.01)and the expression level of its target gene STAT1 was significantly higher than that in the control group(P<0.05),which was in line with expectations.The spleen of the mice in the MRL/lpr group was significantly larger than that of normal mice(P<0.05).The spleen of the mice injected with miR-150-5p agomir group was smaller than that of the NC group,while the spleen of miR-1505p antagomir group was larger,but there was no statistical significance.The number of B cells in the blood and spleen of mice in MRL/lpr group was significantly increased compared with that of normal mice(P<0.001).The number of B cells in the blood of mice in miR-150-5p agomir group was decreased compared with that in NC group(P<0.001),while the number of B cells in miR-150-5p antagomir group was significantly increased(P<0.001).Compared with the control group,the expression levels of IgG,anti-DS-DNA,ANA,IL-6 and IL-12 in the model group were significantly increased(P<0.05),while the expression levels of C3 were significantly decreased(P<0.05).After the intervention of miR-150-5p agomir,the inflammatory response in SLE mice could be alleviated.HE and PAS staining results showed that the renal pathological changes of MRL/lpr mice were alleviated after agomir injection,while antagomir injection could be aggravated to a certain extent.PCR,Western blot and luciferase reporter gene assay results proved that Mir150-5p could regulate the expression of STAT1.Conclusion:miRNA can be involved in the occurrence and development of SLE.In PBMC of SLE patients,miR-150-5p is significantly low in expression,and STAT1 is highly expressed.miR-15 0-5p can promote the degradation of STAT1 by binding to STAT1’s 3’UTR.As a result,STAT1 expression,transcription and translation processes are reduced,thereby inhibiting the activation of B cells and controlling the disease development of SLE. |
