微小RNA-130b-3p通过抑制ERBB2IP和PPARγ表达促进肾小管上皮细胞转分化

Circulating miRNA-130b-3p promotes epithelial-to-mesenchymal transition in renal tubular epithelial cells by inhibiting expression of ERBB2IP and PPARγ

目的 探讨高表达的微小RNA-130b-3p（MiR-130b-3p）参与肾脏损害的可能机制.方法 肾小管上皮细胞（HK-2）分别转染MiR-130b-3p模拟物（mimic）、对照mimic （NC mimic）,并给予10 μg/L TGF-β1刺激.实时定量PCR和Western印迹分别检测转染72 h后细胞Ⅳ型胶原蛋白（CollegenⅣ）、α-平滑肌肌动蛋白（α-SMA）、Ⅰ型胶原蛋白（CollegenⅠ）、E-钙黏蛋白（E-cadherin）mRNA和蛋白表达水平;生物信息学预测MiR-130b-3p潜在靶基因,Western印迹检测潜在靶基因ERBB2IP和PPARγ蛋白表达水平;分别构建含ERBB2IP 3＆#39;端非编码区（3＆#39;-UTR）和PPARγ 3＆#39;-UTR的双荧光素酶报告基因载体,采用双荧光素酶报告基因方法检测靶基因荧光活性.结果 无刺激转染HK-2细胞后MiR-130b-3p mimic组与NC mimic组比较CollegenⅣ、α-SMA、Collegen Ⅰ、E-cadherin mRNA和蛋白表达差异均没有统计学意义（均P＞0.05）.但在TGF-β1刺激后,与NC mimic组比较,MiR-130b-3p mimic组CollegenⅣ、α-SMA、Collegen Ⅰ mRNA和蛋白表达水平均显著上调,E-cadherin的表达降低（均P＜0.05）.与NC mimic组比较,MiR-130b-3p mimic组潜在靶基因ERBB2IP和PPARγ mRNA和蛋白表达水平均下调（均P＜ 0.05）;双荧光素酶报告基因结果显示,与NC mimic组比较,MiR-130b-3p mimic组ERBB2IP荧光素酶活性降低（P＜0.01）,而突变（mut）-MiR-130b-3p mimic组无明显变化（P＞0.05）;与NC mimic＋mut-PPARγ-3＆#39;UTR共转染组比较,MiR-130b-3p＋mut-PPARγ-3＆#39;UTR组PPARγ荧光素酶活性降低,而PPARγ-3＆#39;UTR共转染MiR-130b-3p组PPARγ荧光素酶活性比mut-PPARγ-3＆#39;UTR共转染MiR-130b-3p组进一步降低,差异均有统计学意义（均P＜0.01）.结论 MiR-130b-3p通过直接抑制ERBB2IP和PPARγ的表达而促进肾小管上皮细胞上皮细胞-间充质细胞转分化作用,从而参与狼疮肾炎早期肾脏损害.

Objective To investigate a possible molecular mechanism of MiRNA-130b-3p involved in renal damage.Methods Human renal tubular epithelial cells （HK-2） were transfected with MiR-130b-3p mimics or normal control mimics.Then HK-2 cells were stimulated with 10 μg/L recombinant TGF-β1 for 72 h.After 72 h, the mRNA and protein expression of Collegen Ⅳ, a-smooth muscle actin （α-SMA）, Collegen Ⅰ and E-cadherin were quantified by real-time PCR and Western blotting.The mRNA and protein expression of ERBB2IP and PPARγ were also detected.The reporter plasmids containing ERBB2IP 3-UTR and PPARγ 3＆#39;-UTR were constructed.The activity of ERBB2IP and PPARγ were detected by dual luciferase report system.Results Compared to NC mimic group,transfection of HK-2 cells with MiR-130b-3p mimics resulted in significantly increased expression of mRNA and protein of Collegen Ⅳ, α-SMA, Collegen Ⅰ, and decreased expression of E-cadherin after stimulating by TGF-β1 （all P 〈 0.05）.And MiR-130b-3p mimic could significantly down-regulate the mRNA and protein expression of ERBB2IP and PPARγ in HK-2 cells （all P 〈 0.05） whether in the presence of TGF-β1 or not.The dual luciferase reporter assay showed that MiR-130b-3p induced decreased ERBB2IP 3＆#39;-UTR luciferase activity compared to NC mimic group, but there was no significant difference between NC mimic group and mut-MiR-130b-3p mimic group.MiR-130b-3p mimic＋mut-PPARγ-3＆#39;UTR cotransfection group had lower PPARγ luciferase activity than NC mimic ＋ mut-PPARγ-3＆#39;UTR group , and MiR-130b-3p＋PPARγ-3UTR group got lower further （all P 〈 0.01）.Conclusions MiR-130b-3p promotes epithelial-to-mesenchymal transition （EMT） in renal tubular epithelial cells by directly targeting at the 3＇-UTR of ERBB2IP and PPARγ, which may play an important role in renal damage of early stage lupus nephritis.