Limits...
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.

Show MeSH

Related in: MedlinePlus

Effects of miR-211 over-expression on melanoma cells.A and B: Relative mean cell titers of (A) WM1552C/211(400), WM1552C/211(800), and WM1552C/VO (vector only) cells to that of WM1552C cells and of (B) A375/211(400) and A375/VO (vector only) cells to that of A375 cells. (C) and (D): Cell invasion assays comparing WM1552C to WM1552C/VO, WM1552C/211(400), WM1552C/211(800), and WM1552C/KC KO stable derivatives, as well as to WM1552C/211(800) transfected with the Anti-miR miRNA Inhibiter for hsa-miR-211 [labelled “WM1552C/211(800) + miR-211”] or Negative Control #1 (labelled “WM1552C/211(800) + miR-Scramble”) over 48 hours. Each assay was performed in triplicate. Statistical significance is indicated by an asterisk in the figure pertaining to the experimental group delimited by a bar over the histograms (P-value<0.001). E) The artificial expression of KCNMA1 protein in WM1552C/211(800) cells increases melanoma cell invasiveness. Western blot results show that KCNMA1 protein levels are elevated in transfected cells [“WM1552C/211(800) + KCNMA1 Vector” relative to control cells without KCNMA1 expression vector] (bottom). β-tubulin was used as a load control. Results from the invasion assay illustrate that the KCNMA1 protein expression increased melanoma cell invasiveness (top).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2967468&req=5

pone-0013779-g008: Effects of miR-211 over-expression on melanoma cells.A and B: Relative mean cell titers of (A) WM1552C/211(400), WM1552C/211(800), and WM1552C/VO (vector only) cells to that of WM1552C cells and of (B) A375/211(400) and A375/VO (vector only) cells to that of A375 cells. (C) and (D): Cell invasion assays comparing WM1552C to WM1552C/VO, WM1552C/211(400), WM1552C/211(800), and WM1552C/KC KO stable derivatives, as well as to WM1552C/211(800) transfected with the Anti-miR miRNA Inhibiter for hsa-miR-211 [labelled “WM1552C/211(800) + miR-211”] or Negative Control #1 (labelled “WM1552C/211(800) + miR-Scramble”) over 48 hours. Each assay was performed in triplicate. Statistical significance is indicated by an asterisk in the figure pertaining to the experimental group delimited by a bar over the histograms (P-value<0.001). E) The artificial expression of KCNMA1 protein in WM1552C/211(800) cells increases melanoma cell invasiveness. Western blot results show that KCNMA1 protein levels are elevated in transfected cells [“WM1552C/211(800) + KCNMA1 Vector” relative to control cells without KCNMA1 expression vector] (bottom). β-tubulin was used as a load control. Results from the invasion assay illustrate that the KCNMA1 protein expression increased melanoma cell invasiveness (top).

Mentions: Since the over-expression of KCNMA1 is often associated with both cell proliferation and cell migration/invasion in various cancers [49]–[51], we decided to determine whether the depletion of miR-211 and associated over-expression of KCNMA1 were important for these processes in melanoma cells. We began by comparing the proliferation rates of melanoma cell lines stably transfected with the miR-211 expression cassette with those of untransfected melanoma cells and cell lines transfected with the empty expression vector (Figure 8A) (see Methods), respectively. All miR-211-expressing cultures of WM1552C/211 showed reduced cell counts compared to those of WM1552C beginning at even the first time point (day 4), and the titre continued to fall behind as time progressed. After a 21-day period, WM1552C/211(400) had greater than 30% decrease in cell counts compared to those of WM1552C, while WM1552C/211(800) cultures showed an even greater decrease in cell proliferation. WM1552C/VO cells showed no significant difference in cell proliferation compared to WM1552C. Comparable results were obtained for cell proliferation of A375/211 cell lines, which grew more slowly than untransfected A375 or A375/VO (Figure 8B). These results are consistent with the hypothesis that an important growth stimulatory event in the melanoma cell lines WM1552C and A375 involves the depletion of miR-211 levels—the latter possibly leading to the targeted up-regulation of at least KCNMA1 expression among its target genes.


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)

Effects of miR-211 over-expression on melanoma cells.A and B: Relative mean cell titers of (A) WM1552C/211(400), WM1552C/211(800), and WM1552C/VO (vector only) cells to that of WM1552C cells and of (B) A375/211(400) and A375/VO (vector only) cells to that of A375 cells. (C) and (D): Cell invasion assays comparing WM1552C to WM1552C/VO, WM1552C/211(400), WM1552C/211(800), and WM1552C/KC KO stable derivatives, as well as to WM1552C/211(800) transfected with the Anti-miR miRNA Inhibiter for hsa-miR-211 [labelled “WM1552C/211(800) + miR-211”] or Negative Control #1 (labelled “WM1552C/211(800) + miR-Scramble”) over 48 hours. Each assay was performed in triplicate. Statistical significance is indicated by an asterisk in the figure pertaining to the experimental group delimited by a bar over the histograms (P-value<0.001). E) The artificial expression of KCNMA1 protein in WM1552C/211(800) cells increases melanoma cell invasiveness. Western blot results show that KCNMA1 protein levels are elevated in transfected cells [“WM1552C/211(800) + KCNMA1 Vector” relative to control cells without KCNMA1 expression vector] (bottom). β-tubulin was used as a load control. Results from the invasion assay illustrate that the KCNMA1 protein expression increased melanoma cell invasiveness (top).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0013779-g008: Effects of miR-211 over-expression on melanoma cells.A and B: Relative mean cell titers of (A) WM1552C/211(400), WM1552C/211(800), and WM1552C/VO (vector only) cells to that of WM1552C cells and of (B) A375/211(400) and A375/VO (vector only) cells to that of A375 cells. (C) and (D): Cell invasion assays comparing WM1552C to WM1552C/VO, WM1552C/211(400), WM1552C/211(800), and WM1552C/KC KO stable derivatives, as well as to WM1552C/211(800) transfected with the Anti-miR miRNA Inhibiter for hsa-miR-211 [labelled “WM1552C/211(800) + miR-211”] or Negative Control #1 (labelled “WM1552C/211(800) + miR-Scramble”) over 48 hours. Each assay was performed in triplicate. Statistical significance is indicated by an asterisk in the figure pertaining to the experimental group delimited by a bar over the histograms (P-value<0.001). E) The artificial expression of KCNMA1 protein in WM1552C/211(800) cells increases melanoma cell invasiveness. Western blot results show that KCNMA1 protein levels are elevated in transfected cells [“WM1552C/211(800) + KCNMA1 Vector” relative to control cells without KCNMA1 expression vector] (bottom). β-tubulin was used as a load control. Results from the invasion assay illustrate that the KCNMA1 protein expression increased melanoma cell invasiveness (top).
Mentions: Since the over-expression of KCNMA1 is often associated with both cell proliferation and cell migration/invasion in various cancers [49]–[51], we decided to determine whether the depletion of miR-211 and associated over-expression of KCNMA1 were important for these processes in melanoma cells. We began by comparing the proliferation rates of melanoma cell lines stably transfected with the miR-211 expression cassette with those of untransfected melanoma cells and cell lines transfected with the empty expression vector (Figure 8A) (see Methods), respectively. All miR-211-expressing cultures of WM1552C/211 showed reduced cell counts compared to those of WM1552C beginning at even the first time point (day 4), and the titre continued to fall behind as time progressed. After a 21-day period, WM1552C/211(400) had greater than 30% decrease in cell counts compared to those of WM1552C, while WM1552C/211(800) cultures showed an even greater decrease in cell proliferation. WM1552C/VO cells showed no significant difference in cell proliferation compared to WM1552C. Comparable results were obtained for cell proliferation of A375/211 cell lines, which grew more slowly than untransfected A375 or A375/VO (Figure 8B). These results are consistent with the hypothesis that an important growth stimulatory event in the melanoma cell lines WM1552C and A375 involves the depletion of miR-211 levels—the latter possibly leading to the targeted up-regulation of at least KCNMA1 expression among its target genes.

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