miR-10b通过下调KLF10促进高糖诱导的肾小管上皮细胞EMT

miR-10b promotes high glucose-stimulated epithelial-mesenchymal transition of renal tubular epithelial cells by repressing KLF10 expression

目的:探讨微小RNA-10b(miR-10b)在高糖诱导的肾小管上皮细胞上皮-间充质转化(EMT)中的作用及相关机制。方法:通过RT-qPCR法分析2型糖尿病合并肾纤维化患者肾组织中miR-10b的表达量;利用高糖刺激人肾小管上皮细胞HK-2建立EMT模型,RT-qPCR法检测miR-10b在EMT不同阶段的表达水平。在HK2细胞中分别转染miR-10b模拟物(mimic)和抑制物(inhibitor) 24 h后,再给予高糖刺激,相差显微镜观察细胞形态学改变,Western blot法检测纤连蛋白(fibronectin)和N-钙黏蛋白(N-cadherin)等EMT标志蛋白的表达。通过在线数据库miRBase对miR-10b的作用靶点进行预测,并利用双萤光素酶报告基因实验进行验证。结果:与癌旁正常肾组织相比,2型糖尿病合并肾纤维化患者肾组织中miR-10b的表达明显增加(P<0.01)。在高糖刺激诱导的HK-2细胞EMT中,miR-10b的表达呈现时间依赖性上调(P<0.01)。转染miR-10b inhibitor可抑制高糖诱导的HK-2细胞形态学改变以及EMT标志蛋白fibronectin、SLUG、N-cadherin和SNAI1表达的上调。在线数据库预测KLF10的3’-UTR可以与miR-10b结合,该结果被双萤光素酶报告基因实验证实。同时,KLF10过表达可以逆转高糖诱导的肾小管上皮细胞的形态学改变以及fibronectin和N-cadherin等EMT标志蛋白。进一步研究显示,miR-10b是通过抑制KLF10表达,从而激活TGF-β/Smad3通路发挥作用。结论:miR-10b可通过下调KLF10表达促进高糖诱导的肾小管上皮细胞EMT。

AIM: To explore the effect and the underlying mechanisms of microRNA-10 b(miR-10 b) on high glucose-stimulated epithelial-mesenchymal transition(EMT) of renal tubular epithelial cells. METHODS: The expression level of miR-10 b was examined by RT-qPCR in the kidney tissues of the type 2 diabetes patients with kidney fibrosis. The EMT model of HK-2 cells was induced by high glucose stimulation and the miR-10 b expression in the process was detected by RT-qPCR. The morphological changes of the HK-2 cells were observed using a microscope. EMT markers, such as fibronectin and N-cadherin, were examined by Western blot. The online database predicted that the 3’-UTR of KLF10 bound to miR-10 b and their direct interaction was confirmed by dual luciferase report assay. RESULTS: Compared with the para-carcinoma normal tissues, the expression level of miR-10 b was up-regulated in the tissues of type 2 diabetes patients with kidney fibrosis(P<0.01). In high glucose-stimulated HK-2 cells, the expression level of miR-10 b was increased in a time-dependent manner(P<0.01). miR-10 b inhibitor reversed the morphological changes and the increases expression of the EMT markers including fibronectin, SLUG, N-cadherin and SNAI1 induced by high glucose stimulation. Online database showed miR-10 b was able to bind with the 3’-UTR in the promoter region of KLF10, thus negatively regulating its expression. Meanwhile, over-expression of KLF10 inhibited the EMT induced by high glucose. Inhibition of TGF-β/Smad3 activation was observed during the process of KLF10-repressed EMT. CONCLUSION: miR-10 b promotes high glucose-stimulated epithelial-mesenchymal transition of renal tubular epithelial cells may through repressing KLF10 expression.