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Snail and Slug collaborate on EMT and tumor metastasis through miR-101-mediated EZH2 axis in oral tongue squamous cell carcinoma

View Article: PubMed Central

ABSTRACT

microRNAs(miRNAs) can regulate epithelial-mesenchymal transition (EMT) through transcription factors, however, little is known whether EMT transcription factors can modulate miRNAs and further induce EMT and cancer metastasis. Here we show that overexpression of Snail and Slug leads to a mesenchymal phenotype and morphology and enhances cell invasion along with stem cell properties in squamous cell carcinoma of oral tongue (OTSCC) cells. Repression of miR-101 expression by Snail and Slug is essential for Snail/Slug-induced malignant phenotypes. The suppression of miR-101 subsequently activates EZH2, the sole histone methyltransferase, inducing EMT, migration and invasion of OTSCC cells. Importantly, co-overexpression of Slug and Snail correlates with poor survival and elevated EZH2 expression in two independent patient cohorts of OTSCC specimens. These findings defined a Snail and Slug/miR-101/EZH2 pathway as a novel regulatory axis of EMT-mediated-microRNA signaling.

No MeSH data available.


Related in: MedlinePlus

Snail/Slug-induced EMT generated stem cell-like cells(A) Morphologic change of Cal-27 cells expressing empty vector, Snail, Slug, or Snail/Slug. Scale bar, 100 mm. (B) The results showed that the Snail/Slug-expressing cells exhibited a significant down-regulation of E-cadherin and up-regulation of Vimentin, compared with vector group and Snail group (p < 0.05). Snail/Slug-expressing cells had a significant higher level of Vimentin than Slug group (p < 0.001), however, there was no difference of E-cadherin expression between Snail/Slug group and Slug group (p > 0.05). Error bars represent the mean ± SD of triplicate experiments. (C) Phase contrast images and quantification of mammospheres formation. Scale bar, 100 mm. Error bars represent the mean ± SD of triplicate experiments. (D) FACS analysis of cell-surface markers CD133 and CD44 in cells expressing Snail/Slug or empty vector. Percentages of mean CD133+/CD44+ subpopulation ± SD based on triplicate experiments are indicated.
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Figure 3: Snail/Slug-induced EMT generated stem cell-like cells(A) Morphologic change of Cal-27 cells expressing empty vector, Snail, Slug, or Snail/Slug. Scale bar, 100 mm. (B) The results showed that the Snail/Slug-expressing cells exhibited a significant down-regulation of E-cadherin and up-regulation of Vimentin, compared with vector group and Snail group (p < 0.05). Snail/Slug-expressing cells had a significant higher level of Vimentin than Slug group (p < 0.001), however, there was no difference of E-cadherin expression between Snail/Slug group and Slug group (p > 0.05). Error bars represent the mean ± SD of triplicate experiments. (C) Phase contrast images and quantification of mammospheres formation. Scale bar, 100 mm. Error bars represent the mean ± SD of triplicate experiments. (D) FACS analysis of cell-surface markers CD133 and CD44 in cells expressing Snail/Slug or empty vector. Percentages of mean CD133+/CD44+ subpopulation ± SD based on triplicate experiments are indicated.

Mentions: To further confirm that the effect of Snail or Slug silencing on the decrease of migration and invasion of Cal-27 cells is specific, we stably overexpressed Snail, Slug, or both proteins in Cal-27 cells (Supplementary Figure S2A). Overexpression of Snail or Slug restored migration and invasion, and cells that co-expressed both proteins demonstrated the highest ability for migration and invasion (Supplementary Figure S2B). The mRNA and protein levels of epithelial markers reduced and the mesenchymal markers increased in Snail or Slug-restored cells, compared with control cells, and co-expressed both proteins gained the highest change of these markers (Supplementary Figure S2C). Importantly, we observed that co-expressed both protein cells displayed an elongated fibroblast-like morphology with scattered distribution in culture, whereas control cells retained their cobblestone-like morphology with tight cell–cell adhesion (Figure 3A). The same results were obtained in Tca8113 cells. Then, we applied RT-PCR to examine the mRNA level of E-cadherin and Vimentin in Snail/Slug groups. The results showed that the Snail/Slug-expressing cells exhibited a significant down-regulation of E-cadherin and up-regulation of Vimentin, compared with vector group and Snail group (p < 0.05), Snail/Slug-expressing cells had a significant higher level of Vimentin than Slug group (p < 0.001) and there was no difference of E-cadherin expression between Snail/Slug group and Slug group (p > 0.05, Figure 3B). These data indicated that both co-overexpression of Snail and Slug contributed to the migratory and invasive behaviors and EMT in OTSCC cells.


Snail and Slug collaborate on EMT and tumor metastasis through miR-101-mediated EZH2 axis in oral tongue squamous cell carcinoma
Snail/Slug-induced EMT generated stem cell-like cells(A) Morphologic change of Cal-27 cells expressing empty vector, Snail, Slug, or Snail/Slug. Scale bar, 100 mm. (B) The results showed that the Snail/Slug-expressing cells exhibited a significant down-regulation of E-cadherin and up-regulation of Vimentin, compared with vector group and Snail group (p < 0.05). Snail/Slug-expressing cells had a significant higher level of Vimentin than Slug group (p < 0.001), however, there was no difference of E-cadherin expression between Snail/Slug group and Slug group (p > 0.05). Error bars represent the mean ± SD of triplicate experiments. (C) Phase contrast images and quantification of mammospheres formation. Scale bar, 100 mm. Error bars represent the mean ± SD of triplicate experiments. (D) FACS analysis of cell-surface markers CD133 and CD44 in cells expressing Snail/Slug or empty vector. Percentages of mean CD133+/CD44+ subpopulation ± SD based on triplicate experiments are indicated.
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Figure 3: Snail/Slug-induced EMT generated stem cell-like cells(A) Morphologic change of Cal-27 cells expressing empty vector, Snail, Slug, or Snail/Slug. Scale bar, 100 mm. (B) The results showed that the Snail/Slug-expressing cells exhibited a significant down-regulation of E-cadherin and up-regulation of Vimentin, compared with vector group and Snail group (p < 0.05). Snail/Slug-expressing cells had a significant higher level of Vimentin than Slug group (p < 0.001), however, there was no difference of E-cadherin expression between Snail/Slug group and Slug group (p > 0.05). Error bars represent the mean ± SD of triplicate experiments. (C) Phase contrast images and quantification of mammospheres formation. Scale bar, 100 mm. Error bars represent the mean ± SD of triplicate experiments. (D) FACS analysis of cell-surface markers CD133 and CD44 in cells expressing Snail/Slug or empty vector. Percentages of mean CD133+/CD44+ subpopulation ± SD based on triplicate experiments are indicated.
Mentions: To further confirm that the effect of Snail or Slug silencing on the decrease of migration and invasion of Cal-27 cells is specific, we stably overexpressed Snail, Slug, or both proteins in Cal-27 cells (Supplementary Figure S2A). Overexpression of Snail or Slug restored migration and invasion, and cells that co-expressed both proteins demonstrated the highest ability for migration and invasion (Supplementary Figure S2B). The mRNA and protein levels of epithelial markers reduced and the mesenchymal markers increased in Snail or Slug-restored cells, compared with control cells, and co-expressed both proteins gained the highest change of these markers (Supplementary Figure S2C). Importantly, we observed that co-expressed both protein cells displayed an elongated fibroblast-like morphology with scattered distribution in culture, whereas control cells retained their cobblestone-like morphology with tight cell–cell adhesion (Figure 3A). The same results were obtained in Tca8113 cells. Then, we applied RT-PCR to examine the mRNA level of E-cadherin and Vimentin in Snail/Slug groups. The results showed that the Snail/Slug-expressing cells exhibited a significant down-regulation of E-cadherin and up-regulation of Vimentin, compared with vector group and Snail group (p < 0.05), Snail/Slug-expressing cells had a significant higher level of Vimentin than Slug group (p < 0.001) and there was no difference of E-cadherin expression between Snail/Slug group and Slug group (p > 0.05, Figure 3B). These data indicated that both co-overexpression of Snail and Slug contributed to the migratory and invasive behaviors and EMT in OTSCC cells.

View Article: PubMed Central

ABSTRACT

microRNAs(miRNAs) can regulate epithelial-mesenchymal transition (EMT) through transcription factors, however, little is known whether EMT transcription factors can modulate miRNAs and further induce EMT and cancer metastasis. Here we show that overexpression of Snail and Slug leads to a mesenchymal phenotype and morphology and enhances cell invasion along with stem cell properties in squamous cell carcinoma of oral tongue (OTSCC) cells. Repression of miR-101 expression by Snail and Slug is essential for Snail/Slug-induced malignant phenotypes. The suppression of miR-101 subsequently activates EZH2, the sole histone methyltransferase, inducing EMT, migration and invasion of OTSCC cells. Importantly, co-overexpression of Slug and Snail correlates with poor survival and elevated EZH2 expression in two independent patient cohorts of OTSCC specimens. These findings defined a Snail and Slug/miR-101/EZH2 pathway as a novel regulatory axis of EMT-mediated-microRNA signaling.

No MeSH data available.


Related in: MedlinePlus