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Introduction

Malignant melanoma is the most aggressive neoplasm with severe metastatic potential. In recent decades, the incidence of malignant melanoma has steadily increased. A particularly worrying feature of the tumor is its increasing incidence and its capacity for rapid metastatic spread. microRNAs represent a class of endogenously expressed, small non-coding RNAs that regulate gene expression (1,2). As a consequence of translation, these mRNAs are inhibited or destabilized, resulting in downregulation of the encoded protein. A few microRNAs have been classified as oncogenes or tumor-suppressor genes as their expression is altered in tumors, which in some cases has been shown to contribute to the phenotypes of cancer cells. Recently, the microRNA-34 (miR-34) family was identified as the mediator of tumor suppression by p53 (1). Many reports suggest that the miR-34s contribute to inhibition of invasion or metastasis in various tumors. These facts suggest that miR-34s play an important role as inhibitors of tumor growth. However, the biological characteristics of miR-34s in human malignant melanoma are not well understood (3). In this study, we evaluated the expression of miR-34 family members 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.

Materials and methods

Cell culture

Human melanoma cell lines A375 and G361 were obtained from Dainippon Pharmaceutical Co., Ltd. (Osaka, Japan) and the Health Science Research Resources Bank (Osaka, Japan), respectively. SK-MEL-24 and C32TG were obtained from the American Type Culture Collection (Manassas, VA, USA). These four cell lines were confirmed to have the wild-type p53 gene status (4–6). Cell lines were maintained in Dulbecco’s modified minimal essential medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin.

Quantitative evaluation of miR-34s

Total RNA containing microRNA was extracted using a mirVana™ miRNA Isolation Kit (Applied Biosystems, Foster City, CA, USA) and cDNA was synthesized using a TaqMan® MicroRNA Reverse Transcription Kit (Applied Biosystems). Quantitative reverse transcription PCR (RT-PCR) for miR-34a/b/c and U6 snRNA(RNU6B) was performed according to the manufacturer’s recommendations. The primers for miR-34a (MI0000268), miR-34b (MI0000742), miR-34c (MI0000743) and RNU6B (NR002752) were purchased from TaqMan® MicroRNA Assays (Applied Biosystems). We used TaqMan® Universal PCR Master Mix, No AmpErase® UNG (Applied Biosystems) for the real-time PCR. Real-time RT-PCR assays were run on Thermal Cycler Dice® TP800 (Takara Bio, Inc., Japan) with the comparative ΔCt method (7,8). All samples were assayed in quadruplicate and values were normalized by the respective amounts of RNU6B expression as an endogenous control. The positive control standard was T5, a thrombospondin-2-overexpressing human melanoma cell line established by our laboratory (9).

Growth analyses of the cell lines

The cells were seeded at 1.0×103 cells/well in 2-cm dishes. The cell number was determined with a Coulter Counter (Beckman Coulter, Fullerton, CA). Quadruplicate cultures of each cell line were prepared at all time points (10).

In vitro invasion/migration assays

Cell invasion was assayed in BD BioCoat™ Matrigel™ Invasion Chambers (24-well, 8-μm pore, Becton-Dickinson Labware, Bedford, MA, USA). Control insert chambers were used for migration assays. DMEM supplemented with 5% FBS was used as a chemoattractant. Cells (2.5×103) were suspended in serum-free DMEM and seeded onto the invasion chambers and control chambers. After 24 h of incubation, cells were fixed with methanol and stained with crystal violet for 15 min. Cells remaining on the upper face of the membranes were scraped off and those on the lower face were counted using an inverted microscope. All assays were performed in triplicate. The results were calculated by using the following formula: %Invasion = (mean count of invading cells)/(mean count of migrating cells) ×100.

Statistical analysis

Statistical comparisons of data sets were analyzed by one-way factorial ANOVA and post-hoc test (Dunnett). Data are shown as means ± standard error of the mean (SEM). These analyses were performed using JMP version 8 software (SAS Institute Inc., Cary, NC, USA). P-values <0.05 were considered to denote statistical significance.

Results

Expression of miR-34s in the melanoma cell lines

All four melanoma cell lines showed significant expression of miR-34s as follows – A375: miR-34a 0.6176±0.0295, miR-34b 0.7625±0.0630, miR-34c 0.7877±0.1126; G361: 7.6424±0.2011, 16.4127±0.7376, 22.0332±1.8522; C32TG: 2.1630±0.7064, 2.1091±0.7209, 8.4425±3.3104; SK-MEL-24: 0.3621±0.0559, 2.5659±0.7612, 8.5907±0.5193 (Fig. 1). Significant differences were noted in the expression of each microRNA (ANOVA, p<0.0001 in miR-34a/b/c). Dunnett’s post-hoc test against A375 revealed significant overexpression of miR-34a in G361 (p<0.001) and C32TG (p=0.0302), that of miR-34b in G361 (p<0.001), and that of miR-34c in G361 (p<0.001), C32TG (p=0.0387) and SK-MEL-24 cells (p=0.0351).

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).

Growth characteristics of malignant melanoma cell lines

We examined the growth characteristics of the four cell lines in cell culture conditions (Fig. 2). Their cell doubling times were as follows: A375 23:23, G361 68:24, C32TG 47:22 and SK-MEL-24 67:03 (values shown are h:min).

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).

In vitro invasion/migration assays

To investigate the invasive ability of the four cell lines, Matrigel™ invasion assays were performed (Fig. 3). The percent invasion of each cell line was as follows: A375, 44.00±6.52%; G361, 22.37±2.71%; C32TG, 13.76±2.75%; SK-MEL-24, 45.05±6.71%. These results revealed significant differences (ANOVA, p=0.005). Dunnett’s post-hoc test against A375 showed significant differences in G361 (p=0.0405) and C32TG (p=0.0074).

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).

Discussion

In the present study, we examined the biological role of the miR-34 family in four human malignant melanoma cell lines (A375, G361, C32TG, SK-MEL-24). Real-time PCR revealed that all four melanoma cell lines showed significant expression of the miR-34s, although expression levels of all miR-34s in the A375 cell line were extremely low. Compared with A375, the expression levels of miR-34a in G361 and C32TG cells, miR-34b in the G361 cells, and miR-34c in the G361, C32TG and SK-MEL-24 cell lines were significantly high. The proliferative ability of A375 was higher than that of the other three cell lines. The in vitro invasiveness of A375 and SK-MEL-24 was greater than that of the G361 and C32TG cell lines. These results suggest that overexpression of miR-34a and c suppresses the invasive and generative potential, respectively, of human malignant melanoma.

p53 is activated by the deregulated expression of oncogenes, which induce replication stress and thereby DNA damage (11). Apart from the direct repressive effects of p53 on core promoters, the induction of microRNAs represents an attractive mechanism for the downregulation of proteins observed after p53 activation (12). MicroRNAs form a class of endogenously expressed, small non-coding RNAs with a recently established key role in the post-transcriptional regulation of gene expression (13–15). microRNA family miR-34s, known for their role in the p53 tumor-suppressor network, are controlled in a tissue-specific manner by p53 directly, inducing apoptosis, cell cycle arrest and senescence (1,2,16–24). Several target genes of miR-34s have been identified (18,25,26). The miR-34 family comprises three processed microRNAs that are encoded by two different genes: miR-34a is encoded by its own transcript, whereas miR-34b and miR-34c share a common primary transcript (1).

In malignant melanoma, the interplay between miR-137 and miR-182 was reported to play a key role in the MITF (microphthalmia-associated transcription factor) regulating network, resulting in degradation of the extracellular matrix and controlling migration/invasion ability (3,27,28). However, Bemis et al suggested that there may be more microRNAs regulating MITF (27). Lodygin et al showed that expression of miR-34a is silenced in various tumors including malignant melanoma due to aberrant CpG methylation of the corresponding promoter region (29). Migliore et al demonstrated that reduced expression of miR-34b or miR-34c represents an additional pathway for regulating the expression of the MET oncogene in melanocytic cells (30). It was also reported that miR-34a regulates uveal melanoma cell migration through its target gene, c-Met (31).

Ectopic expression of miR-34a was found to cause cell cycle arrest in the G1 phase (16,19,21). In human colon cancer cells, tumor cells showed signs of senescence after introduction of ectopic miR-34a (22). It is also suggested that miR-34a inhibits cell growth and enhances chemosensitivity, as well as cell cycle and apoptosis regulators, in prostate cancer cell lines (32). miR-34b/c, which is also induced by p53, was able to regulate CpG methylation in oral squamous cell carcinoma and colorectal carcinoma (33,34). It was also reported that miR-34b and c represent novel effectors mediating suppression of such critical components of neoplastic growth as cell proliferation and adhesion-independent colony formation of neoplastic epithelial ovarian cells (18). In this study, miR-34a was inversely correlated with invasiveness, and miR-34c reduced the proliferative potential. Many reports have shown that miR-34b and c have similar biological characteristics. Our results showed that expression levels of miR-34b/c were similar to those in other studies, although statistical differences were larger in miR-34c than b. Consequently, there have been many hypotheses regarding the function of miR-34a/b/c in various tumor types. The biological characteristics of miR-34a/b/c closely resemble each other, but are by no means identical; they are suggested to interact closely and overlap with each other.

We showed herein that the miR-34 family reduced the tumorigenesis of malignant melanoma, although the detailed mechanisms are still unclear. However, there is potential to develop new therapeutic approaches based on microRNA biology. These methods are expected to improve the notably poor prognosis and complete lack of effective standard therapies for malignant melanoma.

References