miR-193b Regulates Mcl-1 in Melanoma - PubMed (original) (raw)

miR-193b Regulates Mcl-1 in Melanoma

Jiamin Chen et al. Am J Pathol. 2011 Nov.

Abstract

MicroRNAs play important roles in gene regulation, and their expression is frequently dysregulated in cancer cells. In a previous study, we reported that miR-193b represses cell proliferation and regulates cyclin D1 in melanoma cells, suggesting that miR-193b could act as a tumor suppressor. Herein, we demonstrate that miR-193b also down-regulates myeloid cell leukemia sequence 1 (Mcl-1) in melanoma cells. MicroRNA microarray profiling revealed that miR-193b is expressed at a significantly lower level in malignant melanoma than in benign nevi. Consistent with this, Mcl-1 is detected at a higher level in malignant melanoma than in benign nevi. In a survey of melanoma samples, the level of Mcl-1 is inversely correlated with the level of miR-193b. Overexpression of miR-193b in melanoma cells represses Mcl-1 expression. Previous studies showed that Mcl-1 knockdown cells are hypersensitive to ABT-737, a small-molecule inhibitor of Bcl-2, Bcl-X(L), and Bcl-w. Similarly, overexpression of miR-193b restores ABT-737 sensitivity to ABT-737-resistant cells. Furthermore, the effect of miR-193b on the expression of Mcl-1 seems to be mediated by direct interaction between miR-193b and seed and seedless pairing sequences in the 3' untranslated region of Mcl-1 mRNA. Thus, this study provides evidence that miR-193b directly regulates Mcl-1 and that down-regulation of miR-193b in vivo could be an early event in melanoma progression.

Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

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Figures

Figure 1

Figure 1

Expression of miR-193b and Mcl-1 in melanoma tissue samples. A: miR-193b expression was measured by Agilent miRNA microarray. The black line indicates the mean value for each group. B: Mcl-1 staining was scored in metastatic melanomas, primary melanomas, and benign nevi. Data shown are mean ± SEM. C: Pearson correlation analysis was performed to determine the relationship between miR-193b expression and Mcl-1 expression. The results show an inverse correlation (r = −0.7 and P < 0.001). *P < 0.0001 was calculated using the independent samples _t_-test.

Figure 2

Figure 2

miR-193b down-regulates Mcl-1 and sensitizes melanoma cells to ABT-737. A: Western blot analysis of Mcl-1 and cleaved PARP expression in Malme-3M, MeWo, SK-MEL-2, and SK-MEL-28 cells transfected with miR-193b or negative control. Cells were transfected with 5 nmol/L miRNA precursor (miR-193b or negative control) and were harvested 72 hours after transfection. B: Western blot analysis of cleaved PARP and Mcl-1 in Malme-3M cells transfected with miR-193b or negative control, as indicated, incubated for 56 hours, and treated with 10 mmol/L ABT-737, as indicated. Cells were harvested 72 hours after transfection. C: Western blot analysis of MeWo and SK-MEL-28 cells, as indicated. The procedure was performed as described in B. D: One microgram of Mcl-1 plasmid (pMcl-1) was cotransfected with 5 nmol/L miRNA precursors, as indicated, and treated with 10 nmol/L ABT-737 as described in B. Gamma tubulin was used as the loading control. Representative data from one of three independent experiments are shown.

Figure 3

Figure 3

miR-193b directly targets the 3′ UTR of Mcl-1. A: miR-193b and its predicted seed binding site in the 3′ UTR of Mcl-1 (top). The underlined 8-nucleotide sequence indicates the miR-193b seed region. Relative luciferase activity is shown for reporter constructs containing 3′ UTR or 3′ UTR M (with deleted seed pairing region) (bottom) in cells transfected with miR-193b or negative control. B: As in A, except reporter constructs carried the indicated fragment of the Mcl-1 3′ UTR (top). Four MREs predicted by RNA22 in the 3′ UTR of Mcl-1 (middle). C: As in A, except reporter constructs carried UTR1 M1 or UTR1 M2 (top). UTR1 M1 contains two point mutations, as indicated in bold italic. UTR1 M2 contains no seed binding region. Renilla luciferase activity was measured 24 hours after transfection using the Dual-Glo luciferase assay system (Promega). Data were normalized to firefly luciferase. Data shown are the mean ± SEM of three replicates and are representative of three independent experiments. *P < 0 0.05 and **P < 0.001 were calculated using the paired samples _t_-test.

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