Biogem, Institute of Genetic Research "Gaetano Salvatore", Ariano Irpino, Italy.
Department of Biomedicine, Aarhus University, Aarhus, Denmark.
J Am Soc Nephrol. 2021 Jun 1;32(6):1339-1354. doi: 10.1681/ASN.2020010031. Epub 2021 Mar 16.
MicroRNAs (miRNAs), formed by cleavage of pre-microRNA by the endoribonuclease Dicer, are critical modulators of cell function by post-transcriptionally regulating gene expression.
Selective ablation of Dicer in AQP2-expressing cells (Dicer mice) was used to investigate the role of miRNAs in the kidney collecting duct of mice.
The mice had severe polyuria and nephrogenic diabetes insipidus, potentially due to greatly reduced AQP2 and AQP4 levels. Although epithelial sodium channel levels were decreased in cortex and increased in inner medulla, amiloride-sensitive sodium reabsorption was equivalent in Dicer mice and controls. Small-RNA sequencing and proteomic analysis revealed 31 and 178 significantly regulated miRNAs and proteins, respectively. Integrated bioinformatic analysis of the miRNAome and proteome suggested alterations in the epigenetic machinery and various transcription factors regulating AQP2 expression in Dicer mice. The expression profile and function of three miRNAs (miR-7688-5p, miR-8114, and miR-409-3p) whose predicted targets were involved in epigenetic control (Phf2, Kdm5c, and Kdm4a) or transcriptional regulation (GATA3, GATA2, and ELF3) of AQP2 were validated. Luciferase assays could not demonstrate direct interaction of AQP2 or the three potential transcription factors with miR-7688-5p, miR-8114, and miR-409-3p. However, transfection of respective miRNA mimics reduced AQP2 expression. Chromatin immunoprecipitation assays demonstrated decreased Phf2 and significantly increased Kdm5c interactions at the gene promoter in Dicer mice, resulting in decreased RNA Pol II association.
Novel evidence indicates miRNA-mediated epigenetic regulation of AQP2 expression.
微小 RNA（miRNAs）由内切核酸酶 Dicer 对前微小 RNA 的切割形成，通过对基因表达的转录后调控，是细胞功能的关键调节因子。
使用在 AQP2 表达细胞中选择性剔除 Dicer（Dicer 小鼠）的方法，研究 miRNA 在小鼠集合管中的作用。
这些小鼠出现严重的多尿和肾性尿崩症，可能是由于 AQP2 和 AQP4 水平大大降低所致。尽管皮质中上皮钠通道水平降低，内髓质中增加，但阿米洛利敏感的钠重吸收在 Dicer 小鼠和对照组中是相等的。小 RNA 测序和蛋白质组学分析分别显示 31 个和 178 个显著调节的 miRNA 和蛋白质。miRNA 组和蛋白质组的综合生物信息分析表明，Dicer 小鼠中表观遗传机制和各种转录因子的改变调节 AQP2 的表达。三个 miRNA（miR-7688-5p、miR-8114 和 miR-409-3p）的表达谱和功能得到了验证，其预测靶标参与了 AQP2 的表观遗传控制（Phf2、Kdm5c 和 Kdm4a）或转录调节（GATA3、GATA2 和 ELF3）。荧光素酶测定未能证明 AQP2 或三个潜在转录因子与 miR-7688-5p、miR-8114 和 miR-409-3p 之间存在直接相互作用。然而，转染相应的 miRNA 模拟物可降低 AQP2 的表达。染色质免疫沉淀试验表明，Dicer 小鼠中基因启动子上的 Phf2 减少，Kdm5c 相互作用显著增加，导致 RNA Pol II 结合减少。
新的证据表明 miRNA 介导的 AQP2 表达的表观遗传调控。
