| Summary: | 碩士 === 國立陽明大學 === 醫學生物技術暨檢驗學系 === 103 === B Cell Activating Factor (BAFF) is expressed primarily by macrophages and dendritic cells. BAFF plays an important role in pathogenesis of autoimmune disease. Serum BAFF levels in patients with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) were increased.
Belimumab, a human monoclonal antibody against BAFF had been used to treat SLE, and RA in the phase II clinical trial. In previous studies, most of the researches focused on down-stream effects rather than the regulation of BAFF expression. Recent researches indicated that microRNAs (miRNAs) are key regulators of diverse biological processes, such as development, tumorigenesis, inflammation, immune response, and metabolism. Therefore, we examined the miRNA target genes for regulation of B cell activating factor (BAFF) expression.
At first, we used microRNA.org, TargetScanMouse 6.2 and DIANA - microT v3.0 to predict miRNA regulation on mouse BAFF gene. We found that mmu-miR-182, mmu-miR-183 and mmu-miR-495 predicted by in all three websites. Studies of miR-495 in autoimmune diseases was less than that of miR-182, and miR-183, therefore we focused on miR-495. In addition we found that the levels of miR-495-3p and BAFF mRNA in spleen and serum were higher in lupus mice than that of healthy mice. The expression of miR-495-3p was slightly positively correlated with mRNA and protein levels of BAFF in serum.
Moreover, the level of miR495-3p was increased by LPS treatment after 3 hours, but decreased after LPS treatment for 6 to 12 hours in RAW264.7 cells. Furthermore, the levels of phospho-ERK, phospho-JNK and phospho-p38 were not affected by miR-495-3p after LPS stimulation in RAW264.7 cells. By the result of luciferase assay, miR495-3p was not mainly binding at the miR495-3p site one of BAFF 3’UTR in LPS-treated or non-LPS-treated RAW264.7 cells, but in IFN-γ-treated RAW264.7 cells miR495-3p was mainly binding at the miR495-3p site one of BAFF 3’UTR. The investigation of miR495-3p binding site in BAFF 3’UTR was needed for further confirmation. These results suggest that miR495-3p might strongly inhibit another negative regulator for BAFF to cause the enhanced expression of BAFF.
Therefore, we used bioinformatics websites to find the target genes of miRNA-495, such as ankyrin repeat domain 13c (Andrd13c), mitogen-activated protein kinase kinase kinase 2 (Map3k2), and phosphatase and tensin homolog (PTEN). In addition, we researched the literature to find the negative regulator of BAFF such as suppressor of cytokine signaling 1 (Socs1), suppressor of cytokine signaling 3 (Socs3), TNF receptor-associated factor 2 (Traf2), TNF receptor-associated factor 3 (Traf3). Finally, we didn’t find down-regulated genes in over-expressed with miR-495 in RAW264.7 cells by qPCR.
In summary, further investigations were required to identify the target genes for regulation of B cell activating factor (BAFF) expression by miR-495.
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