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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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The effect of MITF knock-down on TRPM1 and miR-211 expression in pigmented melanoma cells; SKMEL-28.Relative gene expression levels of MITF, TRPM1, and miR-211 in MITF knock-down cells. Three different doses of MITF siRNA (5 nM, 10 nM and 15 nM) was used to knock-down MITF gene and expression values are normalized to scramble siRNA control. Histograms represent the Ratio of RNA Concentration, as measured by qRT-PCR analysis.
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pone-0013779-g007: The effect of MITF knock-down on TRPM1 and miR-211 expression in pigmented melanoma cells; SKMEL-28.Relative gene expression levels of MITF, TRPM1, and miR-211 in MITF knock-down cells. Three different doses of MITF siRNA (5 nM, 10 nM and 15 nM) was used to knock-down MITF gene and expression values are normalized to scramble siRNA control. Histograms represent the Ratio of RNA Concentration, as measured by qRT-PCR analysis.

Mentions: The gene encoding miR-211 is located within the sixth intron of the TRPM1 gene, which encodes multiple polypeptide isoforms including melastatin-1, a transient receptor potential (TRP) protein family member thought to be a potential suppressor of melanoma metastasis [58]. However, the molecular basis of the tumor suppressor activity of TRPM1 gene is not understood. The transcription factor MITF regulates the expression of TRPM1 gene, where the MITF-binding motif (GCTCACATGT) is located in the TRPM1 promoter [58]. This prompted us to examine whether MITF also might transcriptionally regulate miR-211 expression via the TRPM1 promoter. We found that both TRPM1 and miR-211 transcripts are expressed in pigmented but not in the non-pigmented melanoma cells. To determine whether MITF expression modulates miR-211 expression, we knocked down MITF expression by siRNA in the pigmented melanoma cell line SK-MEL28. Three different doses of siRNA (5 nM, 10 nM and 15 nM) were used, and the knock-down efficiency was measured by qRT-PCR. As expected, the extent of reduction in MITF transcript levels directly correlated with the reduction in TRPM1 and miR-211 transcript levels (Figure 7). In conclusion, the results are consistent with the hypothesis that MITF co-ordinately regulates TRPM1 and miR-211 expression. If true, it raises the possibility that one of the ways MITF might also suppress melanoma metastasis is through its transcriptional activation of miR-211 via the TRPM1 promoter, and the consequent negative post-transcriptional effects of miR-211 on KCNMA1 mRNA.


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)

The effect of MITF knock-down on TRPM1 and miR-211 expression in pigmented melanoma cells; SKMEL-28.Relative gene expression levels of MITF, TRPM1, and miR-211 in MITF knock-down cells. Three different doses of MITF siRNA (5 nM, 10 nM and 15 nM) was used to knock-down MITF gene and expression values are normalized to scramble siRNA control. Histograms represent the Ratio of RNA Concentration, as measured by qRT-PCR analysis.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0013779-g007: The effect of MITF knock-down on TRPM1 and miR-211 expression in pigmented melanoma cells; SKMEL-28.Relative gene expression levels of MITF, TRPM1, and miR-211 in MITF knock-down cells. Three different doses of MITF siRNA (5 nM, 10 nM and 15 nM) was used to knock-down MITF gene and expression values are normalized to scramble siRNA control. Histograms represent the Ratio of RNA Concentration, as measured by qRT-PCR analysis.
Mentions: The gene encoding miR-211 is located within the sixth intron of the TRPM1 gene, which encodes multiple polypeptide isoforms including melastatin-1, a transient receptor potential (TRP) protein family member thought to be a potential suppressor of melanoma metastasis [58]. However, the molecular basis of the tumor suppressor activity of TRPM1 gene is not understood. The transcription factor MITF regulates the expression of TRPM1 gene, where the MITF-binding motif (GCTCACATGT) is located in the TRPM1 promoter [58]. This prompted us to examine whether MITF also might transcriptionally regulate miR-211 expression via the TRPM1 promoter. We found that both TRPM1 and miR-211 transcripts are expressed in pigmented but not in the non-pigmented melanoma cells. To determine whether MITF expression modulates miR-211 expression, we knocked down MITF expression by siRNA in the pigmented melanoma cell line SK-MEL28. Three different doses of siRNA (5 nM, 10 nM and 15 nM) were used, and the knock-down efficiency was measured by qRT-PCR. As expected, the extent of reduction in MITF transcript levels directly correlated with the reduction in TRPM1 and miR-211 transcript levels (Figure 7). In conclusion, the results are consistent with the hypothesis that MITF co-ordinately regulates TRPM1 and miR-211 expression. If true, it raises the possibility that one of the ways MITF might also suppress melanoma metastasis is through its transcriptional activation of miR-211 via the TRPM1 promoter, and the consequent negative post-transcriptional effects of miR-211 on KCNMA1 mRNA.

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