靶向细胞外基质磷酸糖蛋白的microRNA的筛选及鉴定

目的:寻找靶向细胞外基质磷酸糖蛋白(MEPE)基因的微小RNA(miRNA),并检测其对人HeLa细胞内源性Mepe基因表达的影响。方法:通过NCBI检索人源Mepe的3’UTR,利用miRNA预测工具TargetScan预测可能靶向Mepe的所有miRNA,通过双萤光素酶报告基因系统检测miRNA与Mepe3’UTR的结合情况,从而初步筛选出可能靶向Mepe的miRNA;同时,用Western印迹检测miRNA经转染后对Mepe基因表达的影响。结果:利用TargetScan预测出36条可能靶向Mepe的miRNA,根据分值及匹配情况从中挑选出6条进行验证;与转染空载体pGL3-cm的相对荧光素值相比,转染miR-376a的相对荧光素值降低较为明显,而当Mepe3’UTR与miR-376a结合位点突变后,miR-376a不能抑制萤光素酶的活性;Western印迹结果显示miR-376a能明显抑制MEPE的表达。结论:miRNA-376a可能是靶向Mepe基因的miRNA,为进一步研究MEPE的功能奠定了基础。

Identification of the microRNA Targeting Matrix Extracellular Phos phoglycoprotein

Objective: To screen the candidate microRNA(miRNA) targeting matrix extracellular phosphoglycopro tein(MEPE),and analyze the miRNA how to effect MEPE expression in human HeLa cells.Methods: Human Mepe 3’UTR was searched by NCBI website,and TargetScan was used to predict the candidate miRNA targeting Mepe.To test whether these miRNA target Mepe,the binding of miRNA and Mepe 3’UTR were tested by using du al-luciferase assays,MEPE expression was analyzed by Western blotting after the miRNA were transfected to hu man HeLa cells.Results: 36 candidate miRNA targeting Mepe were found,and only 6 predicated miRNA target ing Mepe were selected by context score percentile and consequential pairing.Luciferase analysis showed that the activity of wild type 3’UTR reporter was significantly suppressed by miR-376a,suppression by miR-376a depend ed on the wild type miR-376a complementary sites,and was not longer observed in reporter in which miR-376a complementary sites were mutated.miR-376a could inhibit MEPE expression obviously by Western blotting.Con clusion: Our data strongly suggested that Mepe is a direct target of miR-376a,it is the basis to understand the function of MEPE.