miR-204-5p and miR-3065-5p exert antitumor effects on melanoma cells

Abstract

MicroRNA (miR)-204-5p was previously identified to be downregulated in melanoma compared with melanocytic nevi. This observation prompted a functional study on miR-204-5p and the newly-identified miR-3065-5p, two miRNAs suggested to be tumor-suppressive oncomiRs. Application of miR-204-5p mimics or inhibitors resulted in a decrease or increase, respectively, in melanoma cell proliferation and colony formation. miR-204-5p mimics hindered invasion, whereas miR-204-5p inhibitors stimulated cancer cell migration. Modulation of miR-3065-5p led to a decrease in melanoma cell proliferation, altered cell cycle distribution and increased expression levels of its target genes HIPK1 and ITGA1, possibly due to functional modifications identified in these cells. miR-204-5p and miR-3065-5p demonstrated antitumor capacities that may need to be taken into account in the development of melanoma treatment approaches.

Keywords: cell migration; cell proliferation; melanoma; microRNA-204-5p; microRNA-3065-5p.

Figure 1.

Expression of (A) miR-204-5p and (B) miR-3065-5p in melanocytic nevi, melanoma tissues and the melanoma BRO and SK-MEL1 cell lines. The miR-204-5p level differed between BRO melanoma cells and melanocytic nevi group (*P<0.03). N1-N9 melanocytic nevi group; M1-M12 melanoma group. miR, microRNA.

Figure 2.

Transfection efficiency. The transfection efficiency was determined by quantitative polymerase chain reaction. Cells transfected with (A) miR-204-5p and (B) miR-3065-5p inhibitor displayed elevated levels of HMGA2 mediated by let-7c Anti-miR™ Inhibitor. (C) miR-204-5p and (D) miR-3065-5p mimic application resulted in TWF1 expression decrease by miRNA Mimic miR-1 Positive Control application. *P<0.05 vs. negative control. miR, microRNA; TWF1, Twinfilin actin-binding protein 1; HMGA2; High-mobility group AT-hook 2.

Figure 3.

Cell viability/proliferation assay in melanoma BRO and SK-MEL1 cells following modulation of miR-204-5p and miR-3065-5p levels. MTT assay (96 h) in SK-MEL1 cells following transfection with miR-204-5p and miR-3065-5p (A) inhibitors and (B) mimics revealed decrease of cell viability/proliferation. MTT assay in BRO cells 96 h following transfection with miR-204-5p and miR-3065-5p (C) inhibitors and (D) mimics revealed a stable decrease in cell viability/proliferation. (E) CYQUANT cell proliferation assay in SK-MEL1 cells 96 h following transfection with miR-204-5p mimics. (F) CYQUANT cell proliferation assay in BRO cells 96 h following transfection with miR-204-5p mimics. The nuclei of non-proliferating cells are colored blue, whereas proliferating cells express green fluorescence. miR-204-5p mimic transfection decreased the number of proliferating cells compared with the negative control. *P<0.05 vs. negative control. miR, microRNA.

Figure 4.

Apoptosis and cell cycle assay. (A) miR-204-5p inhibitor transfection does not reduce apoptosis in SK-MEL1 melanoma cells. (B) miR-204-5p mimic transfection does not alter apoptosis in SK-MEL1 melanoma cells. (C) miR-204-5p inhibitor transfection does not modulate apoptosis in BRO melanoma cells. (D) miR-204-5p mimic decreased percentage of alive BRO melanoma cells. (E) Application of miR-3065-5p inhibitor in SK-MEL1 melanoma cells does not reduce the level of apoptosis. (F) Application of miR-3065-5p mimic in SK-MEL1 melanoma cells does not reduce the level of apoptosis. (G) Application of miR-3065-5p inhibitor in BRO melanoma cells does not alter the level of apoptosis. (H) Application of miR-3065-5p mimic in BRO melanoma cells does not alter the level of apoptosis. (I) miR-204-5p inhibitor transfection in SK-MEL1 cells resulted in S-G2-phase-positive cell decrease. (J) miR-204-5p mimic transfection in SK-MEL1 cells resulted in G1-phase-positive cell increase and S-G2-phase-positive cell decrease. (K) miR-204-5p inhibitor transfection in BRO cells resulted in S-G2-phase-positive cell decrease. (L) miR-204-5p inhibitor transfection in BRO cells does not affect the phases of the cell cycle. (M) miR-3065-5p inhibitor transfection in SK-MEL1 cells does not affect the phases of the cell cycle. (N) miR-3065-5p mimic transfection in SK-MEL1 cells resulted in G1-phase-positive cell increase and S-G2-phase-positive cell decrease. (O) miR-3065-5p inhibitor transfection in BRO cells does not modulate the phases of the cell cycle. (P) miR-3065-5p mimic transfection in BRO cells does not affect the phases of the cell cycle. (Q) Histograms of Annexin V/7-AAD assay in BRO melanoma cells. (R) Histograms of cell cycle assay based on propidium iodide staining in SK-MEL1 cells. *P<0.05 between miRNA modulated cells vs. negative control. miR, microRNA; FITC, fluorescein isothiocyanate; 7-AAD, 7-Aminoactinomycin D.

Figure 5.

Cell migration and invasion in BRO melanoma cells. (A) miR-204-5p and miR-3065-5p inhibition promoted BRO melanoma cell migration. (B) Application of miR-3065-5p mimics diminished BRO melanoma cell migration, and application of the miR-204-5p mimic did not exert an effect. (C) miR-3065-5p inhibitor transfection increased BRO melanoma cell invasion. (D) miR-204-5p mimic transfection suppressed BRO melanoma cell invasion, whereas miR-3065-5p mimic application promoted cell invasion. *P<0.05 between microRNA modulated cells vs. negative control. (E) Cell migration assay with BRO melanoma cells following miR-204-5p inhibitor application. (F) Cell invasion assay with BRO melanoma cells following miR-204-5p mimic application. miR, microRNA.

Figure 6.

Colony formation assay in BRO melanoma cells. (A) Application of miR-204-5p and miR-3065-5p inhibitors did not affect the rate of colony formation. (B) Application of miR-204-5p mimic and miR-3065-5p mimics decreased the colony-forming ability *P<0.05 between microRNA modulated cells vs. negative control. (C) BRO melanoma cell colonies visualized following crystal violet staining. A decrease in the number of colonies was observed following the application of miR-204-5p mimics compared with the negative control. miR, microRNA.

Figure 7.

miR-204-5p and miR-3065 target gene expression analysis. (A) miR-204-5p inhibitor application exerted no effect on Bcl-2, SOX4 and TGFβR1 expression in SK-MEL1 cells. (B) miR-204-5p mimics decreased Bcl-2 and TGFβR1 expression in SK-MEL1 cells. (C) miR-204-5p inhibitors decreased Bcl-2 and TGFβR1 expression in BRO melanoma cells. (D) miR-204-5p inhibitors decreased TGFβR1 expression in BRO melanoma cells. (E) miR-3065-5p inhibitors induced HIPK1 and ITGA1 expression in SK-MEL1 melanoma cells. (F) miR-3065 mimics upregulated HIPK1 expression in SK-MEL1 cells. (G) miR-3065-5p inhibitors downregulated HIPK1 and ITGA1 levels in BRO melanoma cells. (H) miR-3065-5p mimics induced HIPK1 downregulation and ITGA1 upregulation in BRO melanoma cells. miR, microRNA; Bcl-2, B-cell lymphoma 2; SOX4, Sex Determining Region Y-Box 4; TGFβR1, Transforming growth factor β receptor 1.