The Intrathymic Mir-548k Modulateds CXCL13Expression In Myasthenia Gravis Patients With Thymic Hyperplasia

Objective：The aim of this study is to explore the microRNA regulation ofCXCL13in thymus of Myasthenia Gravis with thymic Hyperplasia patients byusing the miRNA microarray,bioinformatics methods,QRT-PCR and Luciferaseassays.Methods： Thymic tissues of myasthenia gravis are taken from13myasthenia gravis patients with thymic hyperplasia during thymectomy fromMarch2012to May2013at the Department of Cardiothoracic Surgey,whichbelong to the first Affiliated Hospital of Guangxi Medical University,controlThymic tissues are taken from13age-and sex-matched adults undergoingcardiac surgery in the same time,who have no autoimmune diseases. Total RNAof Thymic tissues are extracted by Trizol and hybridized with the microarray.The miRNAs targeting CXCL13gene-3’untranslated region are predicted byusing Bioinformatics. Quantify the expression of CXCL13mRNA andmicroRNA in thymus tissues. Construct the dual-luciferase reporter assay vector which contains CXCL13gene-3’untranslated region and analyse the miRNAsmodulated CXCL13expression by Luciferase assays.Results：1、The miRNA microarray chip analysis identified33miRNAsdifferentially expressed in thymus hyperplasia tissue of myasthenia graviscompared with control Thymic tissues, miR-548k is one of most down-regulatedmiRNA（s1.982，p＜0.01）.2、By Bioinformatical analysis, we find miR-548kcan target CXCL133′UTR（P＜0.05）.3、QRT-PCR show that The expressionof CXCL13mRNA is upregulated(P<0.01) and miR-548k isdownregulated(P<0.01) in thymus hyperplasia tissue of myasthenia gravispatients and they show a negative correlation（r=-0.919,P<0.01）.4、3’-UTRfragment of CXCL13gene was successfully cloned into thepmiR-RB-REPORTTMvector, which was vertified by XhoI digestion and DNAsequencing.5、Comparing with the control group（1±0.0501，P<0.01）, theluciferase activity of pmiR-RB-REPORTTM-CXCL13-3’UTR treated withmiR-548k mimic（s0.385±0.0156，P<0.01）was decreased to61.5％, preliminaryevidence show that the expression of CXCL13gene was modulated bymiR-548k.Conclusions:1. There is a specific miRNA profile in thymus ofMyasthenia Gravis with thymic Hyperplasia patients.2. our work indicates thatmiR-548k inhibits CXCL13expression by post－transcriptional gene silencingto promote myasthenia gravis development and progression.