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MicroRNA-target pairs in human renal epithelial cells treated with transforming growth factor beta 1: a novel role of miR-382.

ABSTRACT: We reported previously an approach for identifying microRNA (miRNA)-target pairs by combining miRNA and proteomic analyses. The approach was applied in the present study to examine human renal epithelial cells treated with transforming growth factor ?1 (TGF?1), a model of epithelial-mesenchymal transition important for the development of renal interstitial fibrosis. Treatment of human renal epithelial cells with TGF?1 resulted in upregulation of 16 miRNAs and 18 proteins and downregulation of 17 miRNAs and 16 proteins. Of the miRNAs and proteins that exhibited reciprocal changes in expression, 77 pairs met the sequence criteria for miRNA-target interactions. Knockdown of miR-382, which was up-regulated by TGF?1, attenuated TGF?1-induced loss of the epithelial marker E-cadherin. miR-382 was confirmed by 3'-untranslated region reporter assay to target five genes that were downregulated at the protein level by TGF?1, including superoxide dismutase 2 (SOD2). Knockdown of miR-382 attenuated TGF?1-induced downregulation of SOD2. Overexpression of SOD2 ameliorated TGF?1-induced loss of the epithelial marker. The study provided experimental evidence in the form of reciprocal expression at the protein level for a large number of predicted miRNA-target pairs and discovered a novel role of miR-382 and SOD2 in the loss of epithelial characteristics induced by TGF?1.

SUBMITTER: Kriegel AJ

PROVIDER: S-EPMC3001085 | biostudies-literature | 2010 Dec

REPOSITORIES: biostudies-literature

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MicroRNA-target pairs in human renal epithelial cells treated with transforming growth factor beta 1: a novel role of miR-382.

Kriegel Alison J AJ Fang Yi Y Liu Yong Y Tian Zhongmin Z Mladinov Domagoj D Matus Isaac R IR Ding Xiaoqiang X Greene Andrew S AS Liang Mingyu M

Nucleic acids research 20100816 22

We reported previously an approach for identifying microRNA (miRNA)-target pairs by combining miRNA and proteomic analyses. The approach was applied in the present study to examine human renal epithelial cells treated with transforming growth factor β1 (TGFβ1), a model of epithelial-mesenchymal transition important for the development of renal interstitial fibrosis. Treatment of human renal epithelial cells with TGFβ1 resulted in upregulation of 16 miRNAs and 18 proteins and downregulation of 17 ...[more]

PMID: 20716515

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Project description:Epithelial-mesenchymal transition (EMT) occurs in several disease states, including renal fibrosis and carcinogenesis. Myofibroblasts produced from EMT of renal tubular cells are responsible for the deposition of extracellular matrix components in a large portion of renal interstitial fibrosis. Transforming growth factor-beta (TGF-beta) plays an essential role in the EMT of renal tubular cells, but the molecular mechanism governing this process remains largely unknown. In this study, we found that RGC-32 (response gene to complement 32) is critical for TGF-beta-induced EMT of human renal proximal tubular cells (HPTCs). RGC-32 is not normally expressed in the HPTCs. However, TGF-beta stimulation markedly activates RGC-32 while inducing an EMT, as shown by the induction of smooth muscle alpha-actin (alpha-SMA) and extracellular matrix proteins collagen I and fibronectin, as well as the reduction of epithelial marker E-cadherin. TGF-beta function is mediated by several signaling pathways, but RGC-32 expression in HPTCs appears to be mainly regulated by Smad. Functionally, RGC-32 appears to mediate TGF-beta-induced EMT of HPTCs. Blockage of RGC-32 using short hairpin interfering RNA significantly inhibits TGF-beta induction of myofibroblast marker gene alpha-SMA while repressing the expression of E-cadherin. In contrast, overexpression of RGC-32 induces alpha-SMA expression while restoring E-cadherin. RGC-32 also inhibits the expression of another adherens junction protein, N-cadherin, suggesting that RGC-32 alone induces the phenotypic conversion of renal epithelial cells to myofibroblasts. Additional studies show that RGC-32 stimulates the production of extracellular matrix components fibronectin and collagen I. Mechanistically, RGC-32 induces EMT via the activation of other transcription factors such as Snail and Slug. RGC-32 knockdown inhibits the expression of Snail and Slug during TGF-beta-induced EMT. Taken together, our data demonstrate for the first time that RGC-32 plays a critical role in TGF-beta-induced EMT of renal tubular cells.

Project description:The E6 and E7 proteins are the major oncogenic drivers encoded by high-risk human papillomaviruses (HPVs). While many aspects of the transforming activities of these proteins have been extensively studied, there are fewer studies that have investigated how HPV E6/E7 expression affects the expression of cellular noncoding RNAs. The goal of our study was to investigate HPV16 E6/E7 modulation of cellular microRNA (miR) levels and to determine the potential consequences for cellular gene expression. We performed deep sequencing of small and large cellular RNAs in primary undifferentiated cultures of human foreskin keratinocytes (HFKs) with stable expression of HPV16 E6/E7 or a control vector. After integration of the two data sets, we identified 51 differentially expressed cellular miRs associated with the modulation of 1,456 potential target mRNAs in HPV16 E6/E7-expressing HFKs. We discovered that the degree of differential miR expression in HFKs expressing HPV16 E6/E7 was not necessarily predictive of the number of corresponding mRNA targets or the potential impact on gene expression. Additional analyses of the identified miR-mRNA pairs suggest modulation of specific biological activities and biochemical pathways. Overall, our study supports the model that perturbation of cellular miR expression by HPV16 E6/E7 importantly contributes to the rewiring of cellular regulatory circuits by the high-risk HPV E6 and E7 proteins that contribute to oncogenic transformation.ImportanceHigh-risk human papillomaviruses (HPVs) are the causative agents of almost all cervical cancers and many other cancers, including anal, vaginal, vulvar, penile, and oropharyngeal cancers. Despite the availability of efficacious HPV vaccines, it is critical to determine how HPVs cause cancer, as many people remain unvaccinated and the vaccine does not prevent cancer development in individuals who are already infected. Two HPV proteins, E6 and E7, are the major drivers of cancer development, and much remains to be learned about how the expression of these viral proteins reprograms infected cells, ultimately resulting in cancer development. Small, noncoding human RNAs, termed microRNAs (miRs), regulate gene expression and have been implicated in almost all human cancers, including HPV-associated cancers. Our study provides a comprehensive analysis of how E6 and E7 alter the expression of human miRs and how this potentially impacts cellular gene expression, which may contribute to cancer development.

Project description:BACKGROUND:The use of microRNAs in the therapy of kidney disease is hampered by the difficulties in their effective delivery. Micro-vesicles (MVs) are known as natural carriers of small RNAs. Our prior research has demonstrated that MVs isolated from mesenchymal stem cells (MSCs) are capable of attenuating kidney injuries induced by unilateral ureteral obstruction and 5/6 sub-total nephrectomy in mice. The present study aimed to evaluate the effects of miR-34a-5p (miR-34a)-modified MSC-MVs on transforming growth factor (TGF)-?1-induced fibrosis and apoptosis in vitro. METHODS:Bone marrow MSCs were modified by lentiviruses over-expressing miR-34a, from which MVs were collected for the treatment of human Kidney-2 (HK-2) renal tubular cells exposed to TGF-?1 (6?ng/mL). The survival of HK-2 cells was determined using 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) and Annexin V-Light 650/propidium iodide (PI) assays. The expression levels of epithelial markers (tight junction protein 1 [TJP1] and E-cadherin) and mesenchymal markers (smooth muscle actin alpha (?-SMA) and fibronectin) in HK-2 cells were measured using Western blot analysis and an immunofluorescence assay. In addition, changes in Notch-1/Jagged-1 signaling were analyzed using Western blotting. Data were analyzed using a Student's t test or one-way analysis of variance. RESULTS:MiR-34a expression increased three-fold in MVs generated by miR-34a-modified MSCs compared with that expressed in control MVs (P?<?0.01, t?=?16.55). In HK-2 cells, TJP1 and E-cadherin levels decreased to 31% and 37% after treatment with TGF-?1, respectively, and were restored to 62% and 70% by miR-34a-enriched MSC-MVs, respectively. The expression of ?-SMA and fibronectin increased by 3.9- and 5.0-fold following TGF-?1 treatment, and decreased to 2.0- and 1.7-fold after treatment of HK-2 cells with miR-34a-enriched MSC-MVs. The effects of miR-34a-enriched MSC-MVs on epithelial-mesenchymal transition (EMT) markers were stronger than control MSC-MVs. The effects of miR-34a-enriched MSC-MVs on these EMT markers were stronger than control MSC-MVs. Notch-1 receptor and Jagged-1 ligand, two major molecules of Notch signaling pathway, are predicted targets of miR-34a. It was further observed that elevation of Notch-1 and Jagged-1 induced by TGF-?1 was inhibited by miR-34a-enriched MSC-MVs. In addition, TGF-?1 exposure also induced apoptosis in HK-2 cells. Although miR-34a-mofidied MSC-MVs were able to inhibit TGF-?1-triggered apoptosis in HK-2 cells, the effects were less significant than control MSC-MVs (control:TGF-?1: miR-nc-MV:miR-34a-MV?=?1.3:0.6:1.1:0.9 for MTT assay, 1.8%:23.3%:9.4%:17.4% for apoptosis assay). This phenomenon may be the result of the pro-apoptotic effects of miR-34a. CONCLUSIONS:The present study demonstrated that miR-34a-over-expressing MSC-MVs inhibit EMT induced by pro-fibrotic TGF-?1 in renal tubular epithelial cells, possibly through inhibition of the Jagged-1/Notch-1 pathway. Genetic modification of MSC-MVs with an anti-fibrotic molecule may represent a novel strategy for the treatment of renal injuries.

Dataset Information

MicroRNA-target pairs in human renal epithelial cells treated with transforming growth factor beta 1: a novel role of miR-382.

Publications

MicroRNA-target pairs in human renal epithelial cells treated with transforming growth factor beta 1: a novel role of miR-382.

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