miR-451靶向调控肾小球系膜细胞Ywhaz基因的表达

目的探讨miR-451对肾小球系膜细胞Ywhaz基因表达的靶向调控。方法应用生物信息学技术对miR-451的靶基因进行预测,PCR法扩增靶基因3′UTR全长片段,插入荧光素酶报告载体pmiR-RB-REPORTTMvector中,构建荧光素酶报告质粒pmiR-Ywhaz-3′UTR。将miR-451 mimics和NC mimics分别转染高糖培养条件下的小鼠系膜细胞,将miR-451 inhibitor和NC inhibitor分别转染低糖培养条件下的小鼠系膜细胞,采用Real-time RT-PCR和Westernblot法检测miR-451和Ywhaz的表达水平;将miR-451 mimics和NC mimics分别与pmiR-Ywhaz-3′UTR共转染高糖培养条件下的小鼠系膜细胞,miR-451 inhibitor和NC inhibitor分别与pmiR-Ywhaz-3′UTR共转染低糖培养条件下的小鼠系膜细胞,检测各组细胞荧光素酶活性。结果 Ywhaz基因3′UTR区有11个碱基与miR-451种子序列匹配,其中7个碱基呈高度保守性;荧光素酶报告质粒经测序鉴定构建正确;miR-451在miR-451 mimics组呈高表达,在miR-451 inhibitor组表达降低;miR-451可抑制Ywhaz的蛋白表达;荧光素酶活性检测发现,miR-451可通过与Ywhaz3′UTR的结合,抑制Ywhaz的表达。结论 miR-451可直接靶向糖尿病相关基因Ywhaz,抑制其蛋白表达,为进一步研究miR-451调控早期糖尿病肾病发生发展的机制提供了依据。

Targeted modulation of miR-451 on expression of Ywhaz gene in glomerular mesangial cells

Objective To investigate of targeted modulation of miR-451 on expression of Ywhaz gene in glomerular mesangial cells.Methods The potential target gene of miR-451 was predicted by bioinformatic technique,of which the 3′UTR full-length fragment was amplified by PCR and inserted into luciferase reporter vector pmiR-RB-REPORTTM vector to construct recombinant plasmid pmiR-Ywhaz-3′ UTR.Mouse glomerular mesangial cells cultured under high glucose condition were transfected with miR-451 mimics and NC mimics,while those under low glucose condition with miR-451 inhibitor and NC inhibitor,respectively,and determined for expression level of miR-451 by real-time PCR and that of Ywhaz by Western blot.In addition,the mouse glomerular mesangial cells cultured under high glucose condition were cotransfected with pmiR-Ywhaz-3′ UTR,miR-451 mimics and NC mimics,while those under low glucose condition with pmiR-Ywhaz-3′ UTR,miR-451 inhibitor and NC inhibitor,respectively,and determined for luciferase activity.Results The 11 bases in 3′UTR of Ywhaz gene were paired to the seed sequence of miR-451,of which 7 were highly conserved.Sequencing proved that recombinant plasmid pmiR-Ywhaz-3′UTR was constructed correctly.miR-451 was highly expressed in miR-451 mimics groups,of which the expression level decreased in miR-451 inhibitor group.Luciferase activity assay showed that miR-451 inhibited the expression of Ywhaz protein by combination to Ywhaz 3′ UTR.Conclusion The miR-451 directly targeted to the diabetes-related gene Ywhaz and inhibited its expression,which provided a theoretical basis for further study on the mechanisms of miR-451 in regulating the onset and progress of diabetic nephropathy(DN).