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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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Expression of predicted target genes of miR-211 in WM1552C.Histograms of log2 transformed mean expression ratios (fold change) of mRNAs in WM1552C to those in the melanocyte line HEM-l are plotted. The computationally predicted target genes were selected according to criteria described in Methods. Only genes with statistically significant fold change in expression were plotted.
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pone-0013779-g004: Expression of predicted target genes of miR-211 in WM1552C.Histograms of log2 transformed mean expression ratios (fold change) of mRNAs in WM1552C to those in the melanocyte line HEM-l are plotted. The computationally predicted target genes were selected according to criteria described in Methods. Only genes with statistically significant fold change in expression were plotted.

Mentions: Differential expression of miRNAs in melanoma may be mechanistically related to melanoma development, or it may be coincidental. If indeed the depleted miRNAs are biologically relevant, melanoma cells should be enriched for their target transcripts levels relative to their corresponding levels in melanocytes. As the first step to identify such mRNA transcripts, we hybridized cDNAs made from total RNA isolated from the melanoma cell line WM1552C and the melanocyte line HEM-l to Affymetrix expression arrays. We then filtered the hybridization intensity data for differential expression of computationally predicted target transcripts of miR-211 (Figure 4) (see Methods). These experiments revealed 26 putative target transcripts whose expression levels were elevated relative to those in HEM-l.


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)

Expression of predicted target genes of miR-211 in WM1552C.Histograms of log2 transformed mean expression ratios (fold change) of mRNAs in WM1552C to those in the melanocyte line HEM-l are plotted. The computationally predicted target genes were selected according to criteria described in Methods. Only genes with statistically significant fold change in expression were plotted.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0013779-g004: Expression of predicted target genes of miR-211 in WM1552C.Histograms of log2 transformed mean expression ratios (fold change) of mRNAs in WM1552C to those in the melanocyte line HEM-l are plotted. The computationally predicted target genes were selected according to criteria described in Methods. Only genes with statistically significant fold change in expression were plotted.
Mentions: Differential expression of miRNAs in melanoma may be mechanistically related to melanoma development, or it may be coincidental. If indeed the depleted miRNAs are biologically relevant, melanoma cells should be enriched for their target transcripts levels relative to their corresponding levels in melanocytes. As the first step to identify such mRNA transcripts, we hybridized cDNAs made from total RNA isolated from the melanoma cell line WM1552C and the melanocyte line HEM-l to Affymetrix expression arrays. We then filtered the hybridization intensity data for differential expression of computationally predicted target transcripts of miR-211 (Figure 4) (see Methods). These experiments revealed 26 putative target transcripts whose expression levels were elevated relative to those in HEM-l.

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