miR-30b-5p 通过直接靶向 SNAI1 调节糖尿病肾病中的肾上皮-间充质转化。
miR-30b-5p modulate renal epithelial-mesenchymal transition in diabetic nephropathy by directly targeting SNAI1.
机构信息
Department of Nephrology, Shanghai Tongren Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China.
Department of Nephrology, Shanghai Tongren Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China.
出版信息
Biochem Biophys Res Commun. 2021 Jan 8;535:12-18. doi: 10.1016/j.bbrc.2020.10.096. Epub 2020 Dec 29.
OBJECT
Renal tubulointerstitial fibrosis plays a significant role in the development of diabetic nephropathy (DN). SNAI1 is a main activator of epithelial-to-mesenchymal transition (EMT) in the process of fibrosis. This study aimed to investigate the effect of miR-30b-5p targeting SNAI1 on the EMT in DN.
METHODS
Bioinformatics and miRNAs microarray analyses were used to predict the candidate miRNA targeting SNAI1, that is miR-30b-5p. The db/db mice was as DN animal model and renal tissues of mice were stained with PAS. The miR-30b-5p expression in mouse and human renal tissue were examined by quantitative RT-PCR (qRT-PCR) and fluorescence in situ hybridization (FISH), while SNAI1 expression was determined by qRT-PCR and immunohistochemistry. Luciferase reporter gene assay was used to confirm miR-30b-5p directly target 3'-UTR of the SNAI1 mRNA. In vitro, HK-2 cells were treated with high glucose to establish hyperglycemia cell model and transfected with miR-30b-5p mimics to overexpress miR-30b-5p. Expression of miR-30b-5p, SNAI1 and EMT related indicators (E-cadherin, a-SMA and Vimentin) in HK-2 cells under different treatments were determined by qRT-PCR and/or western-blot. In addition, immunofluorescence was performed to evaluate a-SMA expression in HK-2 cells under different treatments.
RESULTS
Bioinformatics analyses revealed miR-30b-5p had complementary sequences with SNAI1 mRNA and the seed region of miR-30b-5p was conserved in human and a variety of animals, including mice. Microarray analysis showed miR-30b expression decreased in DN mice, which was further verified in db/db mice by qRT-PCR and in human DN by FISH. Contrary to miR-30b-5p, SNAI1 expression level was upregulated in db/db mice. Correlation analysis suggested SNAI1 mRNA level was negatively with miR-30b-5p level in renal tissue of db/db mice. Luciferase reporter gene assay confirmed miR-30b-5p directly targeted SNAI1 mRNA. In high glucose induced HK-2 cells, expression levels of miR-30b-5p and E-cadherin were decreased, while SNAI1, a-SMA and Vimentin were increased. Overexpression miR-30b-5p in high glucose induced HK-2 cells could reverse that phenomenon to some extent.
CONCLUSION
These findings suggest that miR-30b-5p play a protective role by targeting SNAI1 in renal EMT in DN.
目的
肾间质纤维化在糖尿病肾病（DN）的发展中起重要作用。SNAI1 是纤维化过程中上皮间质转化（EMT）的主要激活剂。本研究旨在探讨靶向 SNAI1 的 miR-30b-5p 对 DN 中 EMT 的影响。
方法
采用生物信息学和 miRNA 微阵列分析预测靶向 SNAI1 的候选 miRNA，即 miR-30b-5p。使用 db/db 小鼠作为 DN 动物模型，并用 PAS 染色小鼠肾组织。通过定量 RT-PCR（qRT-PCR）和荧光原位杂交（FISH）检测小鼠和人肾组织中 miR-30b-5p 的表达，通过 qRT-PCR 和免疫组化检测 SNAI1 的表达。荧光素酶报告基因检测证实 miR-30b-5p 可直接靶向 SNAI1 mRNA 的 3'-UTR。在体外，用高糖处理 HK-2 细胞建立高糖细胞模型，并转染 miR-30b-5p 模拟物以过表达 miR-30b-5p。通过 qRT-PCR 和/或 Western blot 检测不同处理条件下 HK-2 细胞中 miR-30b-5p、SNAI1 和 EMT 相关指标（E-钙粘蛋白、a-SMA 和波形蛋白）的表达。此外，通过免疫荧光法评估不同处理条件下 HK-2 细胞中 a-SMA 的表达。
结果
生物信息学分析显示 miR-30b-5p 与 SNAI1 mRNA 具有互补序列，miR-30b-5p 的种子区域在人类和多种动物（包括小鼠）中是保守的。微阵列分析显示 miR-30b 在 DN 小鼠中表达降低，qRT-PCR 在 db/db 小鼠中进一步验证，FISH 在人 DN 中验证。与 miR-30b-5p 相反，SNAI1 的表达水平在 db/db 小鼠中上调。相关性分析表明，db/db 小鼠肾组织中 SNAI1 mRNA 水平与 miR-30b-5p 水平呈负相关。荧光素酶报告基因检测证实 miR-30b-5p 可直接靶向 SNAI1 mRNA。在高糖诱导的 HK-2 细胞中，miR-30b-5p 和 E-钙粘蛋白的表达水平降低，而 SNAI1、a-SMA 和波形蛋白的表达水平升高。在高糖诱导的 HK-2 细胞中转染 miR-30b-5p 过表达可在一定程度上逆转这种现象。
结论
这些发现表明，miR-30b-5p 通过靶向 SNAI1 在 DN 中的肾 EMT 中发挥保护作用。
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