The Silencing Effect of MicroRNA 496 and AU-Rich Element Binding Protein Tristetraprolin on the Expression of CTLA-4 Gene Associated with T1D Autoimmune Disease Pathogenesis
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- Category: Vol. 78, December 2010
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The Silencing Effect of MicroRNA 496 and AU-Rich Element Binding Protein Tristetraprolin on the Expression of CTLA-4 Gene Associated with T1D Autoimmune Disease Pathogenesis,HATEM M. SALEH
Abstract
Background: The control of RNA stability is an important process enabling immune cells to rapidly adjust the levels of regulatory factors whose overexpression may be detrimental for the cellular functions. Disruption of this balance results in defective immune responses and autoimmune disorders. Cytotoxic T Lymphocyte Antigen 4 (CTLA4) plays a critical role in the control of T cells expansion and associates with the development of autoimmune diseases.
Objective: To study the role for CTLA4 Silencing by miRNA 496 and Tristetraprolin (TTP) AU-Rich Element Binding Protein in autoimmune disease pathogenesis as in Type 1 Diabetes disease.
Methods: Bioinformatics analysis for the 3'UTR-CTLA4 gene was done by using CLUSTALX, DNA Sequencing was done by for the assessment of 3'UTR-CTLA4 sequence in human. Cloning of 3'UTR-CTLA4 construct into pGL3-Luc-control vector transfected into HEK293 cell line. The Dual Luciferase reporter assay and RNA-Protein immunoprecipi-tation used for studying the RNA stability. Total was RNA isolated and cDNA was synthesized for RT-PCR analysis, Moreover, miRNA 496 detection was performed by using Quantitative TaqMan miRNA assay. MiRNA, RNA interfer-ence, protein analysis and fluorescence microscopy was used to study the expression of CTLA-4 and TTP into single granules (SGs).
Results: It was demonstrated that the expansion of the AU-rich element (ARE) within CTLA4 3'UTR dictates mRNA degradation. The study has further identified that miR-496 bind upstream of the AU-rich region and represses CTLA4 translation. The role of miR-496 in CTLA4 mRNA decay was independent of the ARE binding protein tristetraprolin (TTP).
Conclusion: The miRNA targeting of CTLA4 happens at stress granules and is a critical regulatory step acting in parallel with the ARE-mediated mRNA degradation pathway. This mechanism has consequences for T1D pathogenesis and might contribute to the genetic predisposition of CTLA4 to autoim-mune diseases as in type 1 diabetes disease.