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MicroRNA let-7b targets important cell cycle molecules in malignant melanoma cells and interferes with anchorage-independent growth

Julia Schultz, Peter Lorenz, Gerd Gross, Saleh Ibrahim and Manfred Kunz

Figure 1.

Validation of microRNA expression screening of benign melanocytic nevi and primary malignant melanomas. In validation experiments each sample (from 10 benign nevi and 10 primary melanomas) was tested individually for expression of microRNAs let-7a, let-7b, and let-7d, respectively, using commercially available TaqMan^® MicroRNA Assays. Data are given as relative percent of average signals in benign nevi and primary melanomas. Asterisks indicate statistical significance of differences in microRNA expression between benign nevi and primary melanomas (P < 0.05; Mann-Whitney-U test).

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Figure 2.

Influence of microRNA let-7b on cyclins D1, D3, Cdk4, and cyclin A expression in melanoma cells. (A) SK-Mel-147 or G361 melanoma cells were transiently transfected with 30 nM of let-7b or control (co) precursor microRNA. Total protein extracts of cells were subjected to immunoblotting of cyclins D1, D3, Cdk4, and cyclin A. Immunoblots were re-probed with an anti-tubulin mouse monoclonal Ab to verify equal loading of proteins. (B-D) Immunofluorescence analyses of SK-Mel-147 melanoma cells transfected with 30 nM let-7b or control precursor microRNA. At 36 h after transfection melanoma cells were fixed with paraformaldehyde and stained with anti-cyclin D1 (B), anti-Cdk4 (C), or anti-cyclin D3 (D) antibodies. An Alexa Fluor 488 goat anti-mouse IgG antibody or goat anti-rabbit IgG antibody was used as secondary antibody. Cells were counterstained with DAPI. Upper panel shows microscopic pictures of immunofluorescence staining of one representative experiment ( 100 and 400 magnifications, respectively). Lower panel shows graphic presentation of mean fluorescence intensities of three independent experiments. Asterisks in (B-D) indicate statistical significance of differences in cyclin D1, Cdk4, or cyclin D3 expression in let-7b-transfected cells compared with control precursor microRNA-transfected cells (P < 0.05; Mann-Whitney-U test).

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Figure 3.

Reporter gene analyses using CCND1 and CCNA 3'UTR constructs. SK-Mel-147 melanoma cells were transfected with the pGL3 Promotor Vector (pGL3), constructs containing the 3'UTR of CCND1 (pGL3-CCND1-3'UTR), or 3'UTR of CCNA (pGL3-CCNA-3'UTR), and co-transfected with either anti-let-7b antagomir or left untransfected (Ø). Single asterisks indicate statistical significance of differences in pGL3-CCND1-3'UTR or pGL3-CCNA-3'UTR luciferase activity compared with pGL3 luciferase activity; double asterisks indicate statistical significance of difference in pGL3-CCND1-3'UTR luciferase activity of anti-let-7b-transfected compared with untransfected cells (P < 0.05; Mann-Whitney-U test).

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Figure 4.

Influence of let-7b on cell cycle progression of melanoma cells. SK-Mel-147 melanoma cells were transfected for 48 h with 30 nM let-7b precursor microRNA (Pre-let-7b) or anti-let-7b antagomir. Cell cycle analysis was performed by flow cytometry. Data are given as mean values +/- SD of three independent experiments. Asterisks indicate statistical significance of differences in the number of cells in G0-G1, S, or G2-M between Pre-let-7b- or anti-let7b-transfected versus control transfected cells (P < 0.05; Mann-Whitney-U test).

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Figure 5.

Influence of let-7b on colony formation of melanoma cell in vitro. G361 melanoma cells were transfected for 48 h with 30 nM let-7b or control precursor microRNA and grown in soft agar for 20 days at 37 °C. (A) High-power views ( 100 magnification) of colonies from one representative experiment. (B) Graphic presentation of numbers of colony-forming units from three independent experiments. Data in (B) are given as mean values +/- SD. Asterisk indicates statistical significance of the difference in number of colonies of let-7b-transfected compared with control miRNA-transfected cells (P < 0.05; Mann-Whitney-U test).

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Table 1 - Differentially expressed microRNAs in primary malignant melanomas compared with benign nevi, analyzed by TaqMan^® real-time PCR.

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