Hypoxia-Induced Down-Regulation of microRNA-34a Promotes EMT by Targeting the Notch Signaling Pathway in Tubular Epithelial Cells

PLOS ONE, Dec 2019

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 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/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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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. - 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)


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Rui Du, Wenjuan Sun, Lin Xia, Ali Zhao, Yan Yu, Lijuan Zhao, Hanmin Wang, Chen Huang, Shiren Sun. Hypoxia-Induced Down-Regulation of microRNA-34a Promotes EMT by Targeting the Notch Signaling Pathway in Tubular Epithelial Cells, PLOS ONE, 2012, 2, DOI: 10.1371/journal.pone.0030771