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目的:通过证明miR-30a、miR-30b靶向GW182,探索HeLa细胞中miR-30a、miR-30b及GW182的生物学功能。方法:生物信息学预测分析表明GW182的3’UTR区存在4个保守的miR-30a、miR-30b靶位点,将含有靶位点的序列片段构建到萤光素酶报告载体中,检测miR-30a、miR-30b与靶位点的结合情况;用阳离子脂质体转染GW182siRNA和miR-30a、miR-30b mimics,检测GW182下游功能的变化。结果:miR-30a、miR-30b能够靶向GW182;qPCR及Western印迹证明miR-30a、miR-30b可以在mRNA与蛋白水平上降低GW182的表达,同时影响GW182所参与的miRNA/siRNA对靶基因的抑制作用。结论:GW182是miR-30a、miR-30b的靶基因,揭示了miR-30a、miR-30b通过靶向GW182影响miRNA/siRNA作用途径的新功能。
OBJECTIVE: To explore the biological functions of miR-30a, miR-30b and GW182 in HeLa cells by demonstrating that miR-30a and miR-30b target GW182. METHODS: Bioinformatics prediction analysis showed that there are four conserved miR-30a and miR-30b target sites in the 3’UTR region of GW182, and the sequence fragment containing the target site was constructed into a luciferase reporter vector to detect miR -30a, miR-30b and target sites; GW182 siRNA and miR-30a, miR-30b mimics were transfected with cationic liposomes to detect the function of GW182 downstream. Results: miR-30a and miR-30b could target GW182. QPCR and Western blotting demonstrated that miR-30a and miR-30b could reduce the expression of GW182 at the mRNA and protein levels and at the same time affect the target genes Inhibition. Conclusions: GW182 is a target gene of miR-30a and miR-30b, revealing new functions of miR-30a and miR-30b that affect the miRNA / siRNA pathway by targeting GW182.