Hypoxia-Induced Down-Regulation of microRNA-34a Promotes EMT by Targeting the Notch Signaling Pathway in Tubular Epithelial Cells
et al. (2012) Hypoxia-Induced Down-Regulation of microRNA-34a Promotes EMT by Targeting the Notch Signaling
Pathway in Tubular Epithelial Cells. PLoS ONE 7(2): e30771. doi:10.1371/journal.pone.0030771
Hypoxia-Induced Down-Regulation of microRNA-34a Promotes EMT by Targeting the Notch Signaling Pathway in Tubular Epithelial Cells
Rui Du 0
Wenjuan Sun 0
Lin Xia 0
Ali Zhao 0
Yan Yu 0
Lijuan Zhao 0
Hanmin Wang 0
Chen Huang 0
Shiren Sun 0
Fabio Martelli, Istituto Dermopatico dell'Immacolata-IRCCS, Italy
0 1 Department of Nephrology, Xijing Hospital, Fourth Military Medical University , Xi'an, People's Republic China , 2 State Key Laboratory of Cancer Biology & Xijing Digestive Hospital, Fourth Military Medical University , Xi'an, People's Republic China
Background: Hypoxia-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 Findings: miRNA 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 a-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/Significance: Our 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.
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Funding: This work was supported by grants from National Nature Science Foundation of China (No. 81070570, 81000988, and 81100525). The funders had no
role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
. These authors contributed equally to this work.
MicroRNAs (miRNAs) are a class of non-coding, single-stranded,
small RNA molecules about 1925 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]. It has been suggested that miRNAs are
involved in embryonic development, tumorigenesis, metastasis,
metabolism, and many other physiological and pathological
processes [2]. The biological functions of most miRNAs are not
yet fully understood. Recently, miRNAs were demonstrated to be
involved in the process of epithelialmesenchymal transition (EMT)
by modulation of EMT-related genes. EMT is characterized by the
loss of cell polarity and epithelial surface markers, induction of the
expression of mesenchymal markers, and increased motility and
invasiveness [3]. Several studies have shown that members of the
miR-200 family (e.g., miR-141 and miR-200b) and miR-205 can
prevent transforming growth factor b (TGF-b) induced EMT by
downregulating ZEB1 and ZEB2, the two major transcriptional
repressors of E-cadherin, which is a key marker of epithelial cells [4
6]. miR-192 was also 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]. In human renal biopsies, 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.
Chronic hypoxia is one of the final pathways that lead to end
stage renal failure [9]. Recently, it has been well established that
activation of HIF-1 signaling in renal epithelial cells under low
oxyge (...truncated)