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The regulation of miRNA-211 expression and its role in melanoma cell invasiveness.

Mazar J, DeYoung K, Khaitan D, Meister E, Almodovar A, Goydos J, Ray A, Perera RJ - PLoS ONE (2010)

Bottom Line: KCNMA1 mRNA and protein expression levels varied inversely with miR-211 levels.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.

View Article: PubMed Central - PubMed

Affiliation: Sanford Burnham Medical Research Institute, Orlando, Florida, United States of America.

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.

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Differentially expressed miRNA transcripts in the melanoma cell line WM1552C.Histograms of log2 of mean expression ratios of miRNA levels in WM1552C to that in the untransformed melanocyte cell line HEM-l (control) are plotted as histograms. Asterisks indicate the respective levels of statistical significance, indicated below the diagram.
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pone-0013779-g001: Differentially expressed miRNA transcripts in the melanoma cell line WM1552C.Histograms of log2 of mean expression ratios of miRNA levels in WM1552C to that in the untransformed melanocyte cell line HEM-l (control) are plotted as histograms. Asterisks indicate the respective levels of statistical significance, indicated below the diagram.

Mentions: As the first step in identifying down-regulated miRNAs in human melanoma, we identified significantly differentially expressed miRNA species in the melanoma cell line WM1552C (originally established from a stage 3 skin melanoma of a 72-year-old patient) compared to those in the normal melanocyte cell line HEM-l by hybridization of total RNA samples to miRNA probe arrays (see Methods). Figure 1 lists 24 statistically significant differentially expressed miRNAs, classified into three groups according to their significance levels (P<0.01, 0.02, and 0.05, respectively). 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 (qRT-PCR) analysis on RNA isolated from WM1552C and seven additional non-pigmented melanoma cell lines (see Methods) (Figure 2A), addressing the expression levels of three separate microRNAs: miR-let7a, miR-let7g, which were over-expressed, and miR-211 was down-regulated. Northern blot analysis further confirmed these results (Figure 2B). This consistency provided the opportunity to address the significance of the reduced level of miR-211 in melanoma. In the following sections we focus on miR-211 and its target genes as a model of the role of miRNAs that are down-regulated in melanoma, with the aim of determining the role of their target genes that are thus up-regulated in melanoma. miR-211 showed the most robust and consistent changes in expression levels between melanocytes and non-pigmented melanoma cell lines. Results reported in Figures 1 and 2 implicate several additional miRNAs in melanoma; specific studies related to these miRNAs are beyond the scope of this communication, and will be reported elsewhere.


The regulation of miRNA-211 expression and its role in melanoma cell invasiveness.

Mazar J, DeYoung K, Khaitan D, Meister E, Almodovar A, Goydos J, Ray A, Perera RJ - PLoS ONE (2010)

Differentially expressed miRNA transcripts in the melanoma cell line WM1552C.Histograms of log2 of mean expression ratios of miRNA levels in WM1552C to that in the untransformed melanocyte cell line HEM-l (control) are plotted as histograms. Asterisks indicate the respective levels of statistical significance, indicated below the diagram.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2967468&req=5

pone-0013779-g001: Differentially expressed miRNA transcripts in the melanoma cell line WM1552C.Histograms of log2 of mean expression ratios of miRNA levels in WM1552C to that in the untransformed melanocyte cell line HEM-l (control) are plotted as histograms. Asterisks indicate the respective levels of statistical significance, indicated below the diagram.
Mentions: As the first step in identifying down-regulated miRNAs in human melanoma, we identified significantly differentially expressed miRNA species in the melanoma cell line WM1552C (originally established from a stage 3 skin melanoma of a 72-year-old patient) compared to those in the normal melanocyte cell line HEM-l by hybridization of total RNA samples to miRNA probe arrays (see Methods). Figure 1 lists 24 statistically significant differentially expressed miRNAs, classified into three groups according to their significance levels (P<0.01, 0.02, and 0.05, respectively). 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 (qRT-PCR) analysis on RNA isolated from WM1552C and seven additional non-pigmented melanoma cell lines (see Methods) (Figure 2A), addressing the expression levels of three separate microRNAs: miR-let7a, miR-let7g, which were over-expressed, and miR-211 was down-regulated. Northern blot analysis further confirmed these results (Figure 2B). This consistency provided the opportunity to address the significance of the reduced level of miR-211 in melanoma. In the following sections we focus on miR-211 and its target genes as a model of the role of miRNAs that are down-regulated in melanoma, with the aim of determining the role of their target genes that are thus up-regulated in melanoma. miR-211 showed the most robust and consistent changes in expression levels between melanocytes and non-pigmented melanoma cell lines. Results reported in Figures 1 and 2 implicate several additional miRNAs in melanoma; specific studies related to these miRNAs are beyond the scope of this communication, and will be reported elsewhere.

Bottom Line: KCNMA1 mRNA and protein expression levels varied inversely with miR-211 levels.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.

View Article: PubMed Central - PubMed

Affiliation: Sanford Burnham Medical Research Institute, Orlando, Florida, United States of America.

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.

Show MeSH
Related in: MedlinePlus