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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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KCNMA1 mRNA is a direct target of miR-211.A) Western blot analysis of KCNMA1 protein expression in melanocytes and melanoma cell lines. Lysates were prepared from cultures of cells complementing those analyzed by northern blot in Figure 2B, including HEM-l, WM1552C, RPMI-7951, SK-MEL2, HT-144, and A375 and probed by Western blotting with antibodies against KCNMA1 or β-tubulin. B) Relative expression of KCNMA1 mRNA in WM1552C compared to WM1552C expressing miR-211 [WM1552C/211(400)]. Histograms represent relative quantification ratio (RQ) as measured by qRT-PCR analysis. Assays were performed in triplicate. C) Western blot analysis of KCNMA1 protein expression in WM1552C stable cell lines. Lysates were prepared from cultures of untransfected WM1552C and WM1552C stably transfected with expression vectors containing: no miR (WM1552C/VO), miR-211 [both WM1552C/211(400) and WM1552C/211(800)], and an shRNA against the KCNMA1 transcript (WM1552C/KC KO), respectively, and probed by Western blotting with antibodies against KCNMA1 or β-tubulin. D) Inverse correlation between miR-211 expression and KCNMA1 protein levels. miR-211 mean RQ was measured by quantitative RT-PCR in three different strains: WM1552C/VO (normalization standard), WM1552C/211(400), and WM1552C/211(800); KCNMA1 protein levels were measured from relative fluorescence in western blots normalized against fluorescence intensity in WM1552C/VO and β-tubulin load controls. Error bars are standard errors of mean for mean RQ, and standard deviations of relative fluorescence intensity. E) Anti-miR-211 inhibitor reverses KCNMA1 protein levels in melanocytes. Melanocytes were transfected with anti-miR-211 inhibitors, and KCNMA1 protein expression was measured in transfected cells by western blot analysis using a KCNMA1 antibody (β-tubulin was used as a load control). Derepression of KCNMA1 protein in the transfected cells is shown in the lane marked as MC+Inh. MC and MC+Scr are melanocyte controls. F) Inhibitory effect of miR-211 on mRNA containing the KCNMA1 3′-UTR sequences. An expression plasmid containing the KCNMA1 3′-UTR seed sequence for miR-211 was fused to a lacZ reporter gene (labelled, KCNMA1) such that the lacZ mRNA would contain the KCNMA1 3′-UTR sequences (harbouring the miR-211 target site) and was co-transfected into the melanoma cell line A375 with one of the following synthetic miRNAs: miR-211, miR-16-1, miR-34b, miR-let-7a, miR-CE (cel-miR-67), and no miRNA. Histograms are measurements of β-galactosidase activity at OD420. To directly confirm the importance of the miR-211 seed sequence, a plasmid containing the LacZ gene was fused to a mutant KCNMA1 3′UTR seed sequence (labelled, KCNMA1 Mutant), and the expression vector itself without any 3′-UTR fusion to LacZ (labelled, positive control) were also included. The assays were performed in triplicate. The only sample with statistically significant difference is indicated by an asterisk (Kruskal Wallis test, χ2 = 24.142, P<0.001).
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pone-0013779-g006: KCNMA1 mRNA is a direct target of miR-211.A) Western blot analysis of KCNMA1 protein expression in melanocytes and melanoma cell lines. Lysates were prepared from cultures of cells complementing those analyzed by northern blot in Figure 2B, including HEM-l, WM1552C, RPMI-7951, SK-MEL2, HT-144, and A375 and probed by Western blotting with antibodies against KCNMA1 or β-tubulin. B) Relative expression of KCNMA1 mRNA in WM1552C compared to WM1552C expressing miR-211 [WM1552C/211(400)]. Histograms represent relative quantification ratio (RQ) as measured by qRT-PCR analysis. Assays were performed in triplicate. C) Western blot analysis of KCNMA1 protein expression in WM1552C stable cell lines. Lysates were prepared from cultures of untransfected WM1552C and WM1552C stably transfected with expression vectors containing: no miR (WM1552C/VO), miR-211 [both WM1552C/211(400) and WM1552C/211(800)], and an shRNA against the KCNMA1 transcript (WM1552C/KC KO), respectively, and probed by Western blotting with antibodies against KCNMA1 or β-tubulin. D) Inverse correlation between miR-211 expression and KCNMA1 protein levels. miR-211 mean RQ was measured by quantitative RT-PCR in three different strains: WM1552C/VO (normalization standard), WM1552C/211(400), and WM1552C/211(800); KCNMA1 protein levels were measured from relative fluorescence in western blots normalized against fluorescence intensity in WM1552C/VO and β-tubulin load controls. Error bars are standard errors of mean for mean RQ, and standard deviations of relative fluorescence intensity. E) Anti-miR-211 inhibitor reverses KCNMA1 protein levels in melanocytes. Melanocytes were transfected with anti-miR-211 inhibitors, and KCNMA1 protein expression was measured in transfected cells by western blot analysis using a KCNMA1 antibody (β-tubulin was used as a load control). Derepression of KCNMA1 protein in the transfected cells is shown in the lane marked as MC+Inh. MC and MC+Scr are melanocyte controls. F) Inhibitory effect of miR-211 on mRNA containing the KCNMA1 3′-UTR sequences. An expression plasmid containing the KCNMA1 3′-UTR seed sequence for miR-211 was fused to a lacZ reporter gene (labelled, KCNMA1) such that the lacZ mRNA would contain the KCNMA1 3′-UTR sequences (harbouring the miR-211 target site) and was co-transfected into the melanoma cell line A375 with one of the following synthetic miRNAs: miR-211, miR-16-1, miR-34b, miR-let-7a, miR-CE (cel-miR-67), and no miRNA. Histograms are measurements of β-galactosidase activity at OD420. To directly confirm the importance of the miR-211 seed sequence, a plasmid containing the LacZ gene was fused to a mutant KCNMA1 3′UTR seed sequence (labelled, KCNMA1 Mutant), and the expression vector itself without any 3′-UTR fusion to LacZ (labelled, positive control) were also included. The assays were performed in triplicate. The only sample with statistically significant difference is indicated by an asterisk (Kruskal Wallis test, χ2 = 24.142, P<0.001).

Mentions: If miR-211 targets the KCNMA1 transcript, KCNMA1 protein expression levels should inversely correlate with that of miR-211 expression levels. A western blot analysis of KCNMA1 expression was performed, utilizing the same cell lines previously examined by northern blot (Figure 2B) for KCNMA1 transcript expression. KCNMA1 protein expression was very low in normal melanocytes, but high in all melanoma cell lines (Figure 6A), indicating an inverse correlation of expression between KCNMA1 protein and miR-211.


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)

KCNMA1 mRNA is a direct target of miR-211.A) Western blot analysis of KCNMA1 protein expression in melanocytes and melanoma cell lines. Lysates were prepared from cultures of cells complementing those analyzed by northern blot in Figure 2B, including HEM-l, WM1552C, RPMI-7951, SK-MEL2, HT-144, and A375 and probed by Western blotting with antibodies against KCNMA1 or β-tubulin. B) Relative expression of KCNMA1 mRNA in WM1552C compared to WM1552C expressing miR-211 [WM1552C/211(400)]. Histograms represent relative quantification ratio (RQ) as measured by qRT-PCR analysis. Assays were performed in triplicate. C) Western blot analysis of KCNMA1 protein expression in WM1552C stable cell lines. Lysates were prepared from cultures of untransfected WM1552C and WM1552C stably transfected with expression vectors containing: no miR (WM1552C/VO), miR-211 [both WM1552C/211(400) and WM1552C/211(800)], and an shRNA against the KCNMA1 transcript (WM1552C/KC KO), respectively, and probed by Western blotting with antibodies against KCNMA1 or β-tubulin. D) Inverse correlation between miR-211 expression and KCNMA1 protein levels. miR-211 mean RQ was measured by quantitative RT-PCR in three different strains: WM1552C/VO (normalization standard), WM1552C/211(400), and WM1552C/211(800); KCNMA1 protein levels were measured from relative fluorescence in western blots normalized against fluorescence intensity in WM1552C/VO and β-tubulin load controls. Error bars are standard errors of mean for mean RQ, and standard deviations of relative fluorescence intensity. E) Anti-miR-211 inhibitor reverses KCNMA1 protein levels in melanocytes. Melanocytes were transfected with anti-miR-211 inhibitors, and KCNMA1 protein expression was measured in transfected cells by western blot analysis using a KCNMA1 antibody (β-tubulin was used as a load control). Derepression of KCNMA1 protein in the transfected cells is shown in the lane marked as MC+Inh. MC and MC+Scr are melanocyte controls. F) Inhibitory effect of miR-211 on mRNA containing the KCNMA1 3′-UTR sequences. An expression plasmid containing the KCNMA1 3′-UTR seed sequence for miR-211 was fused to a lacZ reporter gene (labelled, KCNMA1) such that the lacZ mRNA would contain the KCNMA1 3′-UTR sequences (harbouring the miR-211 target site) and was co-transfected into the melanoma cell line A375 with one of the following synthetic miRNAs: miR-211, miR-16-1, miR-34b, miR-let-7a, miR-CE (cel-miR-67), and no miRNA. Histograms are measurements of β-galactosidase activity at OD420. To directly confirm the importance of the miR-211 seed sequence, a plasmid containing the LacZ gene was fused to a mutant KCNMA1 3′UTR seed sequence (labelled, KCNMA1 Mutant), and the expression vector itself without any 3′-UTR fusion to LacZ (labelled, positive control) were also included. The assays were performed in triplicate. The only sample with statistically significant difference is indicated by an asterisk (Kruskal Wallis test, χ2 = 24.142, P<0.001).
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Related In: Results  -  Collection

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

pone-0013779-g006: KCNMA1 mRNA is a direct target of miR-211.A) Western blot analysis of KCNMA1 protein expression in melanocytes and melanoma cell lines. Lysates were prepared from cultures of cells complementing those analyzed by northern blot in Figure 2B, including HEM-l, WM1552C, RPMI-7951, SK-MEL2, HT-144, and A375 and probed by Western blotting with antibodies against KCNMA1 or β-tubulin. B) Relative expression of KCNMA1 mRNA in WM1552C compared to WM1552C expressing miR-211 [WM1552C/211(400)]. Histograms represent relative quantification ratio (RQ) as measured by qRT-PCR analysis. Assays were performed in triplicate. C) Western blot analysis of KCNMA1 protein expression in WM1552C stable cell lines. Lysates were prepared from cultures of untransfected WM1552C and WM1552C stably transfected with expression vectors containing: no miR (WM1552C/VO), miR-211 [both WM1552C/211(400) and WM1552C/211(800)], and an shRNA against the KCNMA1 transcript (WM1552C/KC KO), respectively, and probed by Western blotting with antibodies against KCNMA1 or β-tubulin. D) Inverse correlation between miR-211 expression and KCNMA1 protein levels. miR-211 mean RQ was measured by quantitative RT-PCR in three different strains: WM1552C/VO (normalization standard), WM1552C/211(400), and WM1552C/211(800); KCNMA1 protein levels were measured from relative fluorescence in western blots normalized against fluorescence intensity in WM1552C/VO and β-tubulin load controls. Error bars are standard errors of mean for mean RQ, and standard deviations of relative fluorescence intensity. E) Anti-miR-211 inhibitor reverses KCNMA1 protein levels in melanocytes. Melanocytes were transfected with anti-miR-211 inhibitors, and KCNMA1 protein expression was measured in transfected cells by western blot analysis using a KCNMA1 antibody (β-tubulin was used as a load control). Derepression of KCNMA1 protein in the transfected cells is shown in the lane marked as MC+Inh. MC and MC+Scr are melanocyte controls. F) Inhibitory effect of miR-211 on mRNA containing the KCNMA1 3′-UTR sequences. An expression plasmid containing the KCNMA1 3′-UTR seed sequence for miR-211 was fused to a lacZ reporter gene (labelled, KCNMA1) such that the lacZ mRNA would contain the KCNMA1 3′-UTR sequences (harbouring the miR-211 target site) and was co-transfected into the melanoma cell line A375 with one of the following synthetic miRNAs: miR-211, miR-16-1, miR-34b, miR-let-7a, miR-CE (cel-miR-67), and no miRNA. Histograms are measurements of β-galactosidase activity at OD420. To directly confirm the importance of the miR-211 seed sequence, a plasmid containing the LacZ gene was fused to a mutant KCNMA1 3′UTR seed sequence (labelled, KCNMA1 Mutant), and the expression vector itself without any 3′-UTR fusion to LacZ (labelled, positive control) were also included. The assays were performed in triplicate. The only sample with statistically significant difference is indicated by an asterisk (Kruskal Wallis test, χ2 = 24.142, P<0.001).
Mentions: If miR-211 targets the KCNMA1 transcript, KCNMA1 protein expression levels should inversely correlate with that of miR-211 expression levels. A western blot analysis of KCNMA1 expression was performed, utilizing the same cell lines previously examined by northern blot (Figure 2B) for KCNMA1 transcript expression. KCNMA1 protein expression was very low in normal melanocytes, but high in all melanoma cell lines (Figure 6A), indicating an inverse correlation of expression between KCNMA1 protein and miR-211.

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