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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。
Objective To investigate the regulation of miR-451 on the expression of Ywhaz gene in glomerular mesangial cells. Methods The target gene of miR-451 was predicted by bioinformatics technology. The full-length 3’UTR of target gene was amplified by PCR and inserted into luciferase reporter vector pmiR-RB-REPORTTMvector to construct luciferase reporter plasmid pmiR- Ywhaz-3’UTR. The miR-451 mimics and NC mimics were respectively transfected into mouse mesangial cells under high glucose conditions. The miR-451 inhibitor and NC inhibitor were transfected into mouse mesangial cells under low glucose conditions respectively. Real-time RT The expression of miR-451 and Ywhaz was detected by PCR and Western blotting. The miR-451 mimics and NC mimics were co-transfected with pmiR-Ywhaz-3’UTR into mouse mesangial cells under high glucose conditions. The expression of miR-451 Inhibitors and NC inhibitors were co-transfected with pmiR-Ywhaz-3’UTR into mouse mesangial cells under hypoglycemic conditions. The luciferase activity in each group was measured. Results 11 bases in the 3’UTR region of Ywhaz gene matched the miR-451 seed sequence, of which 7 bases were highly conserved. Luciferase reporter plasmids were constructed correctly by sequencing. MiR-451 was found in miR-451 mimics group MiR-451 could inhibit the expression of Ywhaz protein. Luciferase activity assay showed that miR-451 could inhibit the expression of Ywhaz by binding to Ywhaz 3’UTR. Conclusion miR-451 can directly target diabetes-related gene Ywhaz and inhibit its protein expression, which provides a basis for further study on the mechanism of miR-451 regulating the development and progression of early diabetic nephropathy.