【摘要】 哺乳类miR-145通过作用于MHC II的反式作用因子CIITA对MHC II的表达进行调控。为探讨在黑青斑河鲀中miR-145是否对CIITA基因存在直接调控作用,进而间接调控MHC II的表达,将黑青斑河鲀CIITA的3′非编码区(3′UTR)序列亚克隆至pMIR-Report载体中,通过双荧光素酶报告系统研究两者是否存在直接的靶标作用。结果表明:将miR-145类似物和构建的CIITA3′UTR载体共转染至Hela细胞后,荧光素酶活性没有出现预期的下调,反而出现上调;增大或减少miR-145的转染量,荧光素酶活性的变化幅度也会随之改变。将miR-145类似物和构建的CIITA3′UTR载体共转染至COS-7细胞,荧光素酶的活性没有改变。离体转染miR-145到河鲀原代脾细胞中,检测MHC II基因的表达水平也没有变化,表明miR-145确实对河鲀CIITA没有靶标作用。更多还原

【Abstract】 Studies in human suggested that miR-145 regulated MHC II expression through direct binding to CIITA.Tetraodon nigroviridis CIITA 3′UTR sequence was cloned and constructed into pMIR-Report vector,a dual luciferase reporter assay was applied to see whether miR-145 directly targets CIITAin T.nigroviridis.The results show that the luciferase activity increases after co-transfection Hela cells with miR-145 mimics and CIITA3′UTR.However,there is no significant change in the luciferase activity for the co-transfected COS-7cells.Transfect T.nigroviridis primary spleen cells with miR-145 mimics result in no significant change of MHC II expression.Taken together,these results indicate that miR-145 has no direct targeting effect on T.nigroviridis CIITA gene.更多还原