Study On The Mechanism Of EPSTI1 Promoting Abnormal Activation Of B Cells In Primary Sjogren's Syndrome

Part 1 Analysis of differentially expressed genes in primary Sjogren's syndrome by RNA sequencingBackgroundPrimary Sjogren's syndrome(pSS)is a chronic inflammatory autoimmune disease which is mainly involved in exocrine glands.In addition to dry mouth and dry eyes caused by damaged salivary gland and lacrimal gland,kidney,lung,peripheral and central nervous system can also be involved.The pathogenesis of pSS is unknown,the highlight characteristics of immunology is abnormal activation of B cells.Abnormal activation of B cells leads to high blood immunoglobulin and the production of a large number autoantibodies,about 5%of the patients eventually progress into lymphoma.In recent years,the second-generation sequencing technology develops rapidly(Next-generation sequencing,NGS),RNA-seq technology can obtain almost all transcriptional information of specific tissues or cells in a certain state with a comprehensive and rapid manner.Transcriptome research can be used to study the quantitative analysis and qualitative research of genes in specific tissues or cells.The purpose of this study was to find the differential expression genes by sequencing the peripheral blood B cells transcriptome of pSS patients and healthy control.MethodsIn this study,3 cases of pSS in the department of rheumatism of Peking union medical hospital were enrolled,and 3 healthy volunteers were enrolled in the group.Peripheral blood cells were isolated,and CD19+B cells were extracted by magnetic beads,extracted the total RNA of B cells,and the cDNA library needed for the sequencing of transcriptome was constructed,the sequencing was performed using RNA-seq technology.Through comparative analysis,pSS patients and HC differentially expressed genes were screened out,GO annotation and KEGG pathway were enriched to analyze the possible biological functions of these differentially expressed genes.All pSS patients enrolled in the group conformed to the international classification standard of the American European Consensus Group(AECG)in 2002 and the diagnostic criteria of the American College of Rheumatology(ACR)in 2012.Results1.In the transcriptome analysis,by means of comparative analysis the B cell samples of pSS patients and HC,we found 51 up-regulated differentially expression genes and 22 down-regulated differentially expression genes.The up-regulated genes mainly include IFI44L,IFI44,IFIT1,IFIT1,IFIT3,IFIT2,IRF7,IFI6,ISG15 and other type I IFN related genes.2.GO annotation analysis found that the enrichment significantly differentially expressed genes associated with IFN signaling pathway and regulating pathway,KEGG pathway analysis found that these genes are more focused on microbial infection related signaling pathways,such as influenza A virus,hepatitis c virus(HCV)and measles virus and herpes simplex virus infection.3.In addition to the interferon related genes in up-regulated genes,more important gene is ZNF683 and EPSTI1.ZNF683 played an important role in NKT and T cells differentiation.EPSTI1 can promote Stat 1 and p65 into nuclear and phosphorylation,induce the expression of inflammation related genes related in macrophage.4.Among the down-regulated genes,the key genes were ESR2 and EGR1,ESR2 was the estrogen receptor,and EGR1 had the effect of inhibiting cell growth and promoting cell apoptosis.ConclusionIn the peripheral blood B cells of pSS patients,51 up-regulated differentially expression genes and 22 down-regulated differentially expression genes were selected.These up-regulated differential expression genes were mainly focused on IFN signaling pathways and viral infection pathways.Part 2 The role of gene EPSTI1 in the regulation of B cells abnormal activation in primary Sjogren's syndromeBackgroundSjogren's syndrome is a common autoimmune disease,in recent years,more and more evidence indicated that B cells play a central role in the pathogenesis of pSS.The prominent immunology characterized of pSS is abnormal B cell activation,produced a large number of immunoglobulin and a variety of autoantibodies.Through transcriptome sequencing,we screened 51 up-regulated differentially expression genes and 22 down-regulated differentially expression genes in B cell of primary Sjogren's syndrome.GO annotation analysis found that these differentially expressed genes significantly enriched in associated with IFN signaling pathway and regulating pathway,KEGG pathway analysis found that these genes are more focused on microbial infection related signaling pathways,such as influenza A virus,hepatitis c virus(HCV)and measles virus and herpes simplex virus infection.Interferons alpha and Interferons gamma can promote the secretion of BAFF,resulting in B cell and T cell activation,so we want to study genes besides IFN genes,EPSTI1 came into our line of sight,EPSTI1 are epithelial mesenchymal interacting protein 1,in some malignant tumors(such as breast cancer),EPSTI1 can promote the metastasis and growth of cancer cells,previous studies have also found in pSS peripheral blood PBMC,the expression of EPSTI1 was increased,the latest study in a mouse model found that EPSTI1 can promote Stat 1 and p65 into the nucleus and phosphorylation to promote source of bone marrow of mice macrophage inflammatory related gene expression.In this study,the effects of EPSTI1 on the function of B cells were studied in order to preliminarily explain the mechanism of abnormal activation of B cells.MethodsIn this study,40 cases of pSS in the department of rheumatism of Peking union medical hospital were enrolled,and 40 healthy volunteers were enrolled in the group.Separation of peripheral blood mononuclear cells,use magnetic bead to separate CD19+ B cells,total RNA was extracted,use fluorescence quantitative PCR to detection EPSTI1 RNA relative expression,total protein was extracted,use western blot to detect EPSTIT1 protein expression level,siRNA was transfected into the rest of the B cell,use CCK8 detect cell proliferation and flow cytometry detect cell apoptosis.Collecting supernatant,use ELISA detection immunoglobulins levels.After knock down of EPSTI1,the expression level of protein in the downstream pathway was detected by western blot.ALL pSS patients enrolled in the group conformed to the international classification standard of the American European Consensus Group(AECG)in 2002 and the diagnostic criteria of the American College of Rheumatology(ACR)in 2012.Results1.The RNA relative expression of EPSTI1 in peripheral blood B cells of pSS patients was (1.596 ± 0.1821),significantly higher than HC(0.9312 ± 0.0895)(p=0.0024).After ImageJ grayscale quantitative analysis,the protein relative expression level of EPSTI1 in peripheral blood B cells of pSS patients was(1.768 ± 0.1714),significantly higher than HC(1.000 ±0.0119)(p = 0.0042).2.In the stimulation of IL4+Anti-IgM+CD40L,the proliferation B cell counts in siRNA group was(374800 ± 34310),while there was a decrease in the comparison of the control siRNA group(435700± 38130),there was no statistical difference(p=0.2626).In the stimulation of CpG+CD40L,the proliferation B cell counts in siRNA group was(404900 ± 17220),which was significantly lower than that of the control siRNA group(6524001 23560)(p<0.0001).3.No significant difference was found in the early apoptosis and late apoptosis rates of the Blank group,the siRNA group,and the control siRNA group,either in CpG+CD40L or IL4+ anti-igm +CD40L stimulation.4.In the stimulation of CpG+CD40L,the concentration of IgG in the siRNA group B cells was(611.4 ± 46.76)ng/ml,which was significantly reduced(p=0.0011)compared with the IgG concentration(1026 ± 79.42)in the control siRNA group.There was no significant difference in IgA,IgM concentration between siRNA group and control siRNA group(p=0.7299/p=0.4578).In the stimulation of IL4+Anti-IgM+CD40L,there was no significant difference in IgG,IgA and IgM in the siRNA group compared with the control siRNA group.5.72 h After siRNA transfected in B cell lines Ramous and Raji,while total p65 protein level was equal,the phosphorylation level of p65 was significantly decreased in siRNA group than in control siRNA group,and the level of phosphorylation p65 in siRNA group under the condition of no CpG stimulation is low,with the stimulation of CpG,phosphorylation level p65 was gradually increased,the CpG stimulus peak was at 30 min,fell slightly after 45 min.The levels of phosphorylated p38 and phosphorylation JNK of the siRNA group and the control siRNA group were approximately the same.6.72 h After siRNA transfected in primary B cell,while total p65 protein level was equal,the phosphorylation level of p65 was significantly decreased in siRNA group than in control siRNA group,and the level of phosphorylation p65 in siRNA group under the condition of no CpG stimulation is low,with the stimulation of CpG,phosphorylation level p65 was gradually increased,the CpG stimulus peak was at 20 and 30 min,fell slightly after 45 min.The levels of phosphorylated p38 and phosphorylation JNK of the siRNA group and the control siRNA group were approximately the same.7.In B cells of pSS patients,the p-P65 relative expression was(1.800 ± 0.1080),significantly higher than HC(1.000 ± 0.0913)(p=0.0013),the total IKBa relative expression was(0.5500± 0.06455),significantly reduced than HC(1.075 ± 0.0629)(p=0.0011).8.In the Ramous cell,72 h after transfection of siRNA,the relative expression of total IKBa in the siRNA group was(1.580 ± 0.1772),significantly higher than the control siRNA group(0.5800 ± 0.2010)(p=0.0203).In the primary B cell,the same phenomenon was observed,and the relative expression of total IKBa in the siRNA group was(1.660 ± 0.0510),significantly higher than the control group(1.000 +0.0447)(p<0.0010).ConclusionEPSTI1 level expression was up-regulated in peripheral blood B cells of pSS patients.EPSTI1 induced phosphorylation of p65 through TLR9 pathway,caused by the degradation of IKBa,thereby promoting the proliferation of B cells and the secretion of immunoglobulin.