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目的探讨微小RNA(miR-148a)在巨噬细胞中能否靶向DNA甲基转移酶1(DNMT1)从而调节ATP结合盒转运体G1(ABCG1)的甲基化水平。方法运用生物信息学网站预测DNMT1是miR-148a靶基因,利用双萤光素酶报告基因系统验证miR-148a能否与DNMT1(3’UTR)特异性结合;将miR-148a模拟物转染至THP-1源性巨噬细胞,运用实时荧光定量PCR技术检测DNMT1的mRNA水平,运用Western Blot技术检测DNMT1蛋白表达水平;运用焦磷酸测序技术观察miR-148a能否在巨噬细胞中影响ABCG1甲基化水平。结果 miR-148a能显著降低pmir GLODNMT1载体上的萤火虫萤光素酶活性;miR-148a在巨噬细胞中不能降低DNMT1的mRNA水平,但可以降低DNMT1的蛋白质水平,上述差异均有统计学意义(P<0.05);焦磷酸测序发现在THP-1源性巨噬细胞miR-148a未影响ABCG1的甲基化水平。结论 miR-148a可以与DNMT1(3’UTR)特异性结合并且阻遏DNMT1蛋白质合成,但是在巨噬细胞中不能通过改变DNMT1的蛋白水平而影响ABCG1甲基化水平。
Objective To investigate whether methylated levels of ATP-binding cassette transporter G1 (ABCG1) can be regulated by microRNAs (miR-148a) in macrophages by targeting DNA methyltransferase 1 (DNMT1). Methods The bioinformatics website was used to predict that DNMT1 was the target gene of miR-148a. The dual luciferase reporter gene system was used to verify whether miR-148a could specifically bind to DNMT1 (3’UTR). The miR-148a mimics were transfected into THP-1-derived macrophages, DNMT1 mRNA was detected by real-time fluorescence quantitative PCR and DNMT1 protein was detected by Western Blot. The effect of miR-148a on the expression of ABCG1 in macrophages was detected by pyrosequencing Basic level. Results miR-148a significantly decreased the firefly luciferase activity of pmir GLODNMT1 vector. MiR-148a did not reduce the DNMT1 mRNA level in macrophages, but decreased the protein level of DNMT1. The differences were statistically significant ( P <0.05). Pyrosequencing revealed that miR-148a in THP-1-derived macrophages did not affect the methylation level of ABCG1. Conclusion miR-148a specifically binds DNMT1 (3’UTR) and inhibits DNMT1 protein synthesis. However, it can not affect the level of ABCG1 methylation in macrophages by altering the DNMT1 protein level.