MicroRNA-26a inhibits the growth and invasiveness of malignant melanoma and directly targets on MITF gene

Abstract

Metastatic melanoma is the most aggressive form of skin cancer and is refractory to therapy. MicroRNAs have been recently discovered as novel molecules that provide therapeutic benefits against melanoma. This work aims to examine the effects of miR-26a and let-7a on the growth and invasiveness of malignant melanoma in vitro and in vivo. In addition, we elucidate the mechanism of action by identifying the target gene of miR-26a. Both miR-26a and let-7a inhibited proliferation and invasiveness and halted the cell cycle at the G₁/G₀ phase in SKMEL-28 and WM1552C malignant melanoma cell lines. Moreover, miR-26a potently induced apoptosis and downregulated the expressions of microphthalmia-associated transcription factor (MITF) and MAP4K3 in both cell lines. The luciferase reporter assay demonstrated that miR-26a suppresses MITF expression by binding the 3'-UTR, suggesting that MITF is a bona fide target of miR-26a. SiRNA knockdown of the MITF gene confirmed that miR-26a reduced cell viability and induced apoptosis by regulating MITF. Using a murine model, we also found miR-26a significantly retarded the growth of melanoma tumors in vivo. In conclusion, miR-26a and let-7a suppressed the growth and invasiveness of melanoma cells, suggesting that miR-26a and let-7a may represent novel therapies for malignant melanoma.

Figure 1

The effect of miR-26a and let-7a on the cell viability of SKMEL-28 and WM1552C melanoma cells. Cell viability was determined using the MTT assay after 48 h transfection with transfection reagent alone (control), AllStars negative control siRNA (100 nM), miR-26a mimics, and let-7a mimics at a final concentration of 50 or 100 nM. Each experiment was repeated six times and the results are presented as mean±S.D. Asterisks indicate a significant difference (P<0.05) compared with control groups.

Figure 2

MiR-26a inhibited cell migration and invasion. (a and b) After 48 h transfection of negative control microRNA mimics or miR-26a mimics in SKMEL-28 cells at the final concentration of 50 or 100 nM, the cell migration assay was performed as described in the Materials and Methods section. The area of the wound was evaluated under a microscope (×100) and determined using the Image J software (National Institutes of Health, Bethesda, MD, USA). The inhibition of cell migration was measured with the following formula: the percent of healing area=(wound area at 0 h–wound area at 24 h)/(wound area at 0 h). SKMEL-28 (c) and WM1552C (d) melanoma cells were seeded into chambers with serum-free medium after transfection of miR-26a or let-7a at a concentration of 100 nM for 48 h. The chambers were inserted into a 24-well plate with medium containing FBS, and the reduced cell invasion ability was detected by the number of cells located on the bottom side of the chamber observed by microscope (×400) after 24 h incubation. The relative invasive index of each group is presented in a bar graph (e). Experiments were repeated three times independently and the results are presented as the mean±S.D. Asterisks indicate a significant difference (P<0.05) compared with control groups.

Figure 3

Cell cycle perturbation was measured by flow cytometry in SKMEL-28 (a) and WM1552C (b) melanoma cells. After transfection with miR-26a or let-7a (100 nM), cells were stained with PI (50 μg/ml) and analyzed by flow cytometry. The percentage of cells in G₀/G₁, S, and G₂/M phases was calculated using the C Flow Plus Analysis Software (Accuri Cytometers Inc., Ann Arbor, MI, USA) and summarized in the table. The histogram is representative of three independent experiments. Asterisks indicate a significant difference (P<0.05) compared with control groups.

Figure 4

MiR-26a induced apoptosis in SKMEL-28 (a) and WM1552C (b) melanoma cells. Cells were transfected with 50 or 100 nM of miR-26a, negative control microRNA, or transfection reagent alone for 48 h. Cells were then stained with Annexin V-FITC and PI and the fluorescence intensity was measured by flow cytometry. Asterisks indicate a significant difference (P<0.05) from control groups.

Figure 5

MiR-26a induced apoptosis by directly targeting the MITF gene. Cells were transfected with miR-26a (100 nM) or negative control for 48 h. The expressions of two hypothetical target genes, MITF and MAP4K3, were detected by western blot in SKMEL-28 (a) and WM1552C (b) melanoma cell lines. The binding sites of miR-26a on MITF 3′-UTR sequence were predicted using microRNA.org (c). Luciferase reporter vectors containing MITF 3′-UTR or empty vectors were used. Relative luciferase activity was significantly decreased with miR-26a co-transfection compared with the negative control for the reporter vector containing the MITF 3′-UTR but not for the empty vector (d). SEAP luminescence was used for normalization. Error bars represent S.D. of three replicates (P-value <0.01). (e–g) Knockdown of MITF reduced cell viability and induced apoptosis. (e) SKMEL-28 and WM1552C were transfected with siMITF-a or siMITF-b (40 nM) or negative control for 72 h, and the whole-cell extracts were subjected to western blot. (f) Cells were transfected with siMITF-a or siMITF-b at the concentration of 40 or 80 nM for 72 h and the cell viability was examined by MTT assay. Asterisks indicated the significant difference compared with control groups (P<0.05). (g) Cells were transfected with siMITFs (40 nM) or negative control for 72 h and then were stained with Annexin V-FITC and PI, and the fluorescence intensity was measured by flow cytometry (P<0.05).

Figure 6

The effect of miR-26a on tumor growth in mice. B16-F10 mouse melanoma cells were transfected with miR-26a (50 or 100 nM) or negative control for 48 h, and 2×10⁵ transfected cell were subcutaneously implanted into the hint flank of male C57BL/6 mice (n=8) to produce tumors. Tumors were measured in 72 h intervals for 16 days. The representative tumors from different groups during the treatment course are shown in graph (a). Tumor volume was calculated with the following formula: (length x width²)/2. The tumor growth curve versus time course was plotted (b). Data are represented as mean±S.D. *Results comparing miR-26a transfection group and control groups at day 10, 13, and 16 were statistically significant (P<0.05).