MicroRNA-target pairs in human renal epithelial cells treated with transforming growth factor beta 1: a novel role of miR-382 - PubMed (original) (raw)

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

Alison J Kriegel et al. Nucleic Acids Res. 2010 Dec.

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 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 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 TGFβ1, attenuated TGFβ1-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 TGFβ1, including superoxide dismutase 2 (SOD2). Knockdown of miR-382 attenuated TGFβ1-induced downregulation of SOD2. Overexpression of SOD2 ameliorated TGFβ1-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 TGFβ1.

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Figures

Figure 1.

Figure 1.

TGFβ1 had widespread effects on miRNA expression in human renal epithelial cells. HK2 cells were treated with vehicle or 3 ng/ml of TGFβ1 for 24 h. (A) 33 of the 376 miRNAs examined with real-time qPCR array were differentially expressed. n = 3. See ‘Materials and Methods’ section for the criteria of differential expression. (B) Verification of the result of miRNA expression profiling. RNA samples used for the individual Taqman qPCR analysis were independent of those used in the high-throughput profiling with the SYBR Green qPCR array. n = 3 for qPCR array, n = 4 for Taqman qPCR; *significantly different from vehicle (P < 0.05 for Taqman qPCR; see ‘Materials and Methods’ section for the criteria of differential expression for qPCR array).

Figure 2.

Figure 2.

Verification of proteomic results. HK2 cells were treated as described in Table 1. Protein samples used for the western blot analysis were independent of those used for the proteomic analysis. TPM1, tropomyosin 1; SOD2, superoxide dismutase 2. The gel shown for TPM1 was a time course analysis. Only the 48-h time point was plotted in the bar graph for comparison with the proteomic data. n = 4 for proteomics, n = 3 for western blot; *significantly different from vehicle (P < 0.05 for western blot; see ‘Materials and Methods’ section for the criteria of differential expression for the proteomic analysis).

Figure 3.

Figure 3.

miR-382 targeted SOD2 and contributed to TGFβ1-induced loss of epithelial characteristics in human renal epithelial cells. (A) TGFβ1 induced loss of epithelial characteristics in HK2 cells as indicated by typical changes in cell morphology and a suppression of E-cadherin mRNA, an epithelial marker. TGFβ1 was used at 3 ng/ml for 48 h. N = 6, *P < 0.05. (B) Knockdown of miR-382. HK2 cells were treated with vehicle or TGFβ1 in the presence of LNA anti-miR-382 or LNA scrambled control anti-miR (100 nM). n = 6, *P < 0.05 vs. no treatment; #P < 0.05 versus TGFβ1 plus control anti-miR. (C) Knockdown of miR-382 attenuated the suppression of E-cadherin mRNA by TGFβ1. n = 6–9, *P < 0.05 versus control anti-miR. (D) miR-382 interacted with the 3′-UTR of SOD2 in Hela cells. Hela cells were transfected with luciferase reporter constructs containing a 3′-UTR segment of SOD2 or a mutated (SOD2-mut) or partially deleted (SOD2-del) segment. The mutations and deletion were described in the text. The effect of anti-miR-382 (100 nM) compared to control, scramble anti-miR was shown. n = 4, *P < 0.05 versus control anti-miR. (E) miR-382 interacted with 3′-UTR of SOD2 in HK2 cells. HK2 cells were transfected with luciferase reporter constructs as described above. The effect of pre-miR-382 (100 nM), a miR-382 mimic, compared with control, scramble pre-miR was shown. n = 4, *P < 0.05 versus control pre-miR. (F) SOD2 expression was suppressed by TGFβ1, but partially restored by knockdown of miR-382. miR-382 in TGFβ1-treated HK2 cells was knocked down with LNA anti-miR (see Figure 3B). n = 6, *P < 0.05 vs. control anti-miR.

Figure 4.

Figure 4.

SOD2 was protective against TGFβ1-induced loss of epithelial characteristics. (A) Overexpression of SOD2 with a Myc-DDK-tagged plasmid (pSOD2). HK2 cells were not transfected (NT) or were transfected with pSOD2 or an empty vector (2 µg for each 3.5-cm dish). Western blot was performed 24 h later using an SOD2 antibody. (B) Overexpression of SOD2 attenuated TGFβ1-induced downregulation of E-cadherin. n = 5, *P < 0.05.

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