Overexpression of the miR-34 family suppresses invasive growth of malignant melanoma with the wild-type p53 gene

Abstract

Malignant melanoma is the most aggressive neoplasm, with severe metastatic potential. microRNAs represent a class of endogenously expressed, small non-coding RNAs that regulate gene expression. As a consequence, the translation of these mRNAs is inhibited or they are destabilized resulting in downregulation of the encoded protein. The microRNA-34 (miR-34) family, which comprises three processed microRNAs (miR-34a/b/c) was identified as the mediator of tumor suppression by p53. Many reports suggest that the miR-34s contribute to the inhibition of invasion or metastasis in various tumor types. In this study, we evaluated the expression of the miR-34 family in four human melanoma cell lines (A375, G361, C32TG and SK-MEL-24) which have the wild-type p53 gene using real-time reverse transcription PCR. We also examined their generative and invasive characteristics using the cell proliferation assay and the invasion/migration assay, respectively. All four melanoma cell lines showed significant expression of miR-34s - A375: miR-34a 0.6176, miR-34b 0.7625, miR-34c 0.7877; G361: 7.6424, 16.4127, 22.0332; C32TG: 2.1630, 2.1091, 8.4425; SK-MEL-24: 0.3621, 2.5659, 8.5907. The cell doubling times of these cell lines in h:min were as follows: A375 23:23, G361 68:24, C32TG 47:22 and SK-MEL-24 67:03. The in vitro generation times of G361 and SK-MEL-24, which showed increased expression of miR-34c, were significantly shorter than A375 with decreased expression of miR-34c (p=0.0063, ANOVA). Invasion (%) of the four cell lines was as follows: A375 44.0%, G361 22.4%, C32TG 13.8% and SK-MEL-24 45.0%. In vitro invasiveness of G361 and C32TG, which showed increased expression of miR-34a, was significantly suppressed (p= 0.005, ANOVA). These results suggest that overexpression of miR-34a and c suppresses invasive and generative potentials, respectively, in human malignant melanoma.

Figure 1.

Gene expression levels of miR-34a/b/c evaluated by real-time PCR. Values were normalized by the respective amounts of RNU6B expression as an endogenous control. There was a significant difference among the four cell lines (n=4, p<0.0001, ANOVA). Dunnett’s post-hoc test showed significant differences against A375 (^*p<0.05, ^***p<0.001).

Figure 2.

Cell proliferation assay. Cells were seeded at 1.0×10³ cells/well in 6-well plates. The cell doubling times of these cell lines were as follows: A375 23:23, G361 68:24, C32TG 47:22 and SK-MEL-24 67:03 (values shown are h:min).

Figure 3.

In vitro invasion/migration assays. There was a significant difference among the four cell lines (n=3, p=0.005, ANOVA). Dunnett’s post-hoc test showed significant differences between A375 and G361 and C32TG (^*p<0.05).