Abstract
The immediate molecular mechanisms behind invasive melanoma are poorly understood. Recent studies implicate microRNAs (miRNAs) as important agents in melanoma and other cancers. To investigate the role of miRNAs in melanoma, we subjected human melanoma cell lines to miRNA expression profiling, and report a range of variations in several miRNAs. Specifically, compared with expression levels in melanocytes, levels of miR-211 were consistently reduced in all eight non-pigmented melanoma cell lines we examined; they were also reduced in 21 out of 30 distinct melanoma samples from patients, classified as primary in situ, regional metastatic, distant metastatic, and nodal metastatic. The levels of several predicted target mRNAs of miR-211 were reduced in melanoma cell lines that ectopically expressed miR-211. In vivo target cleavage assays confirmed one such target mRNA encoded by KCNMA1. Mutating the miR-211 binding site seed sequences at the KCNMA1 3′-UTR abolished target cleavage. KCNMA1 mRNA and protein expression levels varied inversely with miR-211 levels. Two different melanoma cell lines ectopically expressing miR-211 exhibited significant growth inhibition and reduced invasiveness compared with the respective parental melanoma cell lines. An shRNA against KCNMA1 mRNA also demonstrated similar effects on melanoma cells. miR-211 is encoded within the sixth intron of TRPM1, a candidate suppressor of melanoma metastasis. The transcription factor MITF, important for melanocyte development and function, is needed for high TRPM1 expression. MITF is also needed for miR-211 expression, suggesting that the tumor-suppressor activities of MITF and/or TRPM1 may at least partially be due to miR-211's negative post transcriptional effects on the KCNMA1 transcript. Given previous reports of high KCNMA1 levels in metastasizing melanoma, prostate cancer and glioma, our findings that miR-211 is a direct posttranscriptional regulator of KCNMA1 expression as well as the dependence of this miRNA's expression on MITF activity, establishes miR-211 as an important regulatory agent in human melanoma.
Highlights
Melanoma, a cancer of the pigment-producing cells in the skin epidermis, can be highly metastatic, and malignant melanomas are relatively resistant to standard chemotherapy [1]
To address whether the differential miRNA expression levels observed with WM1552C varied among other established melanoma cell lines, we performed quantitative reverse transcriptase mediated polymerase chain reaction analysis on RNA isolated from WM1552C and seven additional non-pigmented melanoma cell lines (Figure 2A), addressing the expression levels of three separate microRNAs: miR-let7a, miR-let7g, which were over-expressed, and miR-211 was down-regulated
We focus on miR-211 and its target genes as a model of the role of miRNAs that are downregulated in melanoma, with the aim of determining the role of their target genes that are up-regulated in melanoma. miR211 showed the most robust and consistent changes in expression levels between melanocytes and non-pigmented melanoma cell lines
Summary
A cancer of the pigment-producing cells in the skin epidermis, can be highly metastatic, and malignant melanomas are relatively resistant to standard chemotherapy [1]. The exact molecular mechanisms that lead to melanoma are complex and poorly understood [3,4,5,6], and may involve both mutagenic DNA lesions and epigenetic misregulation [7,8,9,10,11]. The 9p21 site harbors the tumor suppressor gene INK4a and accompanies additional inactivating mutations that lead to the constitutive activation of genes such as BRAF [16,17]. The activation of BRAF alone may be insufficient to cause metastatic melanoma, but additional mutagenic or epigenetic events such as the inactivation of tumor suppressor genes, e.g., Pten [18], may be important. Noteworthy is the recent demonstration of abnormally high oncogenic potentials of single melanoma cells [22], emphasizing the need for better understanding the molecular mechanisms of melanoma progression
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