MicroRNA Profiling And Functional Study In Primary Sjogren’s Syndrome

BackgroudPrimary Sjogren’s syndrome(pSS) is a chronic autoimmune disease with a broad clinical spectrum that characiterized of lymphocyte proliferation and progressing organ-specific exocrinopathy to systemic manifestations. The pathogenesis of organs involved in pSS we could see lymphocytes infiltration, lots of autoantibodies detected in serum and accompanied with hyperglobulinemia, indicated immunocyte function disorder. MicroRNAs(miRNAs) as small, single-stranded noncoding Ring NAs, many of which have been highly conserved throughout evolution, is known to regulate cellular processes such as growth, development, aging and apoptosis, and associated with human diseases. The miRNAs have multiple regulating potential to broadly influence human immunocytes, and could control the development and function of innate and adaptive immune response by regulating target mRNA expression, and involved in the pathogenesis of autoimmune diseases. In pSS the immunocytes aggregation as the peripheral blood mononuclear cell (PBMC), is the essential target to exploring pathogenesis of the disease. To date, little is known about the roles of miRNAs in pSS pathogenesis and few studies had been published about it. Based on this point, this study was comparing the PBMC of pSS patients with healthy controls (Health Control, HC) to explore which miRNA expression profiling changes and impacts on the significance for the pSS. Furthermore, the dual-luciferase reporter transfection assay was used to analyze the miRNAs on auto-antigens SSA/Ro, SSB/La regulations, aimed at discovering a set of pSS-related miRNAs to investigate their roles in the pathogenesis of pSS.ObjectiveOur aim was to analysis microRNA expression profile in PBMC of patients with pSS, searching for the special miRNAs associated with pSS and investigate the relative expression of special miRNAs of relevance to pSS clinial spectrum. Furthermore, to explore miRNAs which regulate the expression of antigen of Ro52(52KD-Ro/TRIM21), Ro60(60KD-Ro/TROVE2)and SSB.Methods (1) Using the Exiqon miRNA expression profile chip contains1896mature human miRNAs and146human-related virus miRNAs, the expression of miRNAs in pSS and HC PBMC was detected. Hierarchical clustering analysis was applied for determining pSS differentially expressed micrornas spectrum, and the method of quantitive Real-time PCR (qPCR) was applied for validation.(2) The expression level of miR-181a, miR-155and miR-146a was detected by qPCR method in larger sample size, including33cases of patients with pSS and10cases of healthy controls. We analysised the expression levels of those miRNAs with the pSS patients’organ involvement condition, disease activity index and laboratory tests (IgG, ESR, hsCRP and the titre of ANA).(3) Based on the result of miRNA chip profile, miRecords database and Targetscan software was applicated to predict miRNAs which may target genes of SSA and SSB. We built targeted gene3’ UTR region expression vector and detected regulation effects identified by dual luciferase reporter assay.Results(1) The miRNA profiling microarray results shown that a set of pSS-specific miRNAs expression spectrum which contain382differentially expressed miRNAs, of which202miRNAs upregulated and180miRNAs downregulated.(2) miR-181was detected upregulated in pSS PBMC by qPCR method in an expanded sample size and the relative expression to HC is2.81, with statistical significant difference (P<0.05). The analysis of spectrum correlation with clinical profiles shown that miR-181a was positively related to the titre of ANA, miR-155possibly associated with organ involvement, miR-146a is weak positively related to the ESR.(3) We successfully built recombinant plasmid vector which contained TRIM21, TROVE2-2SSB and3’ UTR region respectively. The result of dual luciferase reporter assay shown that miR-1207, miR-4695regulated TRIM21and miR-229regulated SSB, the luciferase activity in each control group was47.3%,53%and47.3%respectively. MiRNA-181a was dectected possible regulation effect on TRIM21, SSB and the luciferase activity in the control group was75.4%and75.5%respectively.Conclusion The research results shown that the pSS PBMC have a specific miRNA expression profile. Overexpression of miR-181a was detected in pSS PBMC, which was positively correlated with the titer of ANA. MiR-181a, miR-1207, miR-4695and miR- 229was confirmed to have regulation effect on SSA/SSB3’UTR region which was probably involved in process of pathophysiological.