Sfmbt2¹⁰th Intron Hosted MiR-466a-3p Are Important Epigenetic Regulator Of Nfat5Signaling, Osmoregulation And Urine Concentration In Mice

miR-466a-3p and many of its close relatives are miRNAs derived from a single miRNA cluster hosted by the10th intron of the Polycomb group gene Sex combs on the midleg with four MBT domains-2(Sfmbt2) on mouse chromosome2(hence also called Chromosome2miRNA cluster, C2MC). C2MC miRNAs share a high degree of sequence similarity, especially among the-3p sequences. Bioinformatic analyses and our previous studies indicate that C2MC miRNAs were highly hypertonic stress responsive and might participate in the post-transcriptional regulation of osmoregulatory transcription factor OREBP/Nfat5in the kidney. In current study, we investigated the roles of miR-466a-3p and its close relatives in the posttranscriptional regulation of Nfat5, urine concentration, renal osmoregulation and osmoadaptation.Through AVP exposure in mIMCD3and water deprivation in WT FVB mice, we examined the time course expression of miR-466(a/b/c/e/p)-3p and miR-200b-3p Through overexpression or inhibition of miR-466a-3p in mIMCD3cells, we examined how miR-466a-3p affected the expression of OREBP/Nfat5. The renal cortical and medullary expression of miR-466a-5p/3p, OREBP/Nfat5and a few of the transcriptional target genes of OREBP/Nfat5in transgenic mice overexpressing miR-466a-3p were analyzed by real-time RT-PCR. After that, water intake, urine excretion, urine osmolality of the WT, transgenic8-10weeks male mice were analyzed. The morphology of renal tissues for8-10weeks old male WT and transgenic mice as determined by hematoxylin and eosin staining.In the renal medulla of FVB mice that were water deprived, significant down-regulations in medullary miR-466(a/b/c/e/p)-3p were observed at8-h, which sustained at least to16-h and returned to the normal levels by24-h. On the other hand exposure of mIMCD3cells to ddAVP time dependently down-regulated the expression of miR-200b-3p and miR-466(a/b/c/e/p)-3p. In cultured mIMCD3cells, we further showed that miR-466a-3p was capable of regulating Nfat5 post-transcriptionally,through interacting with the corresponding MREs on the3’ UTR. We found that transgenic renal overexpression of miR-466a-3p led to a significant downregulation of osmoregulation-associated genes including Nfat5, Ar,Smit, Aqp2, UtA1, UtA2, UtA3and Sgk1. miR-466a-3p-overexpressing transgenic mice developed modest polydipsia, and polyuria. Menawhile Hemotoxylin and eosin staining showed disturbed morphology of the renal medulla of in transgenic mice as compared with that of the WT mice.We concluded that miR-466a-3p and its close relatives were the important regulators of Nfat5, urine concentration, renal osmoregulation and osmoadaptation.