Abstract
BackgroundHypoxia-induced renal tubular cell epithelial–mesenchymal transition (EMT) is an important event leading to renal fibrosis. MicroRNAs (miRNAs) are small non-coding RNA molecules that bind to their mRNA targets, thereby leading to translational repression. The role of miRNA in hypoxia-induced EMT is largely unknown.Methodology/Principal FindingsmiRNA profiling was performed for the identification of differentially expressed miRNAs in HK-2 cells under normal and low oxygen, and the results were then verified by quantitative real time RT-PCR (qRT-PCR). The function of miRNAs in hypoxia-induced renal tubular cell EMT was assessed by the transfection of specific miRNA inhibitors and mimics. Luciferase reporter gene assays and western blot analysis were performed to validate the target genes of miR-34a. siRNA against Jagged1 was designed to investigate the role of the miR-34a-Notch pathway in hypoxia induced renal tubular cell EMT. miRNA-34a was identified as being downregulated in hypoxic renal tubular epithelial cells. Inhibition of miR-34a expression in HK-2 cells, which highly express endogenous miR-34a, promoted a mesenchymal phenotype accompanied by reduced expression of the epithelial marker Z0-1, E-cadherin and increased expression of the mesenchymal markers α-SMA and vimentin. Conversely, miR-34a mimics effectively prevented hypoxia-induced EMT. Transfection of miRNA-34a in HK-2 cells under hypoxia abolished hypoxia-induced expression of Notch1 and Jagged1 as well as Notch downstream signals, such as snail. Western blot analysis and luciferase reporter gene assays showed direct evidence for miR-34a targeting Notch1 and Jagged1. siRNAs against Jagged1 or Notch1 effectively prevented miR-34a inhibitor-induced tubular epithelial cell EMT.Conclusions/SignificanceOur study provides evidence that the hypoxia-induced decrease of miR-34a expression could promote EMT in renal tubular epithelial cells by directly targeting Notch1 and Jagged1, and subsequently, Notch downstream signaling.
Highlights
MicroRNAs are a class of non-coding, single-stranded, small RNA molecules about 19–25 nucleotides in length, which negatively regulate gene expression at the post-transcriptional level through nucleotide base pairing between complementary sequences of miRNAs and 39-untranslated regions (39UTR) of messenger RNAs [1]
Several studies have shown that members of the miR-200 family and miR-205 can prevent transforming growth factor b (TGF-b) induced epithelial–mesenchymal transition (EMT) by downregulating ZEB1 and ZEB2, the two major transcriptional repressors of E-cadherin, which is a key marker of epithelial cells [4,5,6]. miR-192 was found to repress the E-Box repressors ZEB1 and ZEB2 in tubular epithelial cells and increase collagen 1-a2 production in mesangial cells [7,8]
We asked whether miRNAs could induce EMT under hypoxia since hypoxia is a key factor in the process of EMT and fibrosis [10]
Summary
MicroRNAs (miRNAs) are a class of non-coding, single-stranded, small RNA molecules about 19–25 nucleotides in length, which negatively regulate gene expression at the post-transcriptional level through nucleotide base pairing between complementary sequences of miRNAs and 39-untranslated regions (39UTR) of messenger RNAs (mRNAs) [1]. MiR-192 was found to repress the E-Box repressors ZEB1 and ZEB2 in tubular epithelial cells and increase collagen 1-a2 production in mesangial cells [7,8]. Low expression of miR-192 correlated with tubulointerstitial fibrosis and low estimated GFR [8]. These data suggested that some miRNA species may play important roles in tubular epithelial cell EMT and renal fibrosis. Hypoxia-induced renal tubular cell epithelial–mesenchymal transition (EMT) is an important event leading to renal fibrosis. The role of miRNA in hypoxia-induced EMT is largely unknown
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