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Regulation of epithelial plasticity by miR-424 and miR-200 in a new prostate cancer metastasis model.

Banyard J, Chung I, Wilson AM, Vetter G, Le Béchec A, Bielenberg DR, Zetter BR - Sci Rep (2013)

Bottom Line: DU145-LN4 showed increased cell-cell adhesions, higher expression of multiple epithelial markers, such as E-cadherin, EpCAM and cytokeratin 18, and reduced expression of mesenchymal markers such as vimentin.Ectopic transient and stable miR-424 expression induced EMT, with reduced epithelial marker expression and increased cell scattering.We show that this cellular plasticity can be mediated through the combined action of miR-424 and the miR-200 family.

View Article: PubMed Central - PubMed

Affiliation: 1] Vascular Biology Program, Boston Children's Hospital, Karp Family Research Laboratories, 300 Longwood Ave., Boston, MA 02115 [2] Department of Surgery, Harvard Medical School, Boston, MA 02115.

ABSTRACT
Using an in vivo cycling strategy, we selected metastatic cancer cells from the lymph nodes (LN) of mice bearing orthotopic DU145 human prostate tumors. Repeated rounds of metastatic selection (LN1-LN4) progressively increased the epithelial phenotype, resulting in a new model of tumor cell mesenchymal-epithelial transition (MET). DU145-LN4 showed increased cell-cell adhesions, higher expression of multiple epithelial markers, such as E-cadherin, EpCAM and cytokeratin 18, and reduced expression of mesenchymal markers such as vimentin. The MET in DU145-LN4 cells was accompanied by increased expression of the miR-200 family, and antimiRs to miR-200c and miR-141 induced an EMT. MET also correlated with the loss of miR-424. Ectopic transient and stable miR-424 expression induced EMT, with reduced epithelial marker expression and increased cell scattering. Our model provides evidence for spontaneous MET in vivo. We show that this cellular plasticity can be mediated through the combined action of miR-424 and the miR-200 family.

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MiRNA-424 regulates the epithelial phenotype.(A) Progressive downregulation of miR-424 expression in the DU145-LN cell series was measured by real time RT- PCR. (B) DU145-LN4 cells were transfected with two different control premiRs (left and right panels) or premiR-424 (center panel). PremiR-424 induced EMT-like changes, as observed by reduced cell-cell interactions in phase-contrast microscopy, and decreased E-cadherin and EpCAM levels in immunostaining. (C) Western blot analysis of DU145-LN4 cells treated with premiR controls, premiR-424 or mock transfected, collected at 6 and 21 days shows premiR-424 inhibited E-cadherin and EpCAM expression and increased vimentin levels. Actin shown as loading control. (D) Stable overexpression of miR-424 in DU145-LN4 cells resulted in loss of cell-cell contacts as shown by phase contrast microscopy (scale bar 100 μm). DU145-LN4 vector control (v3), and two miR-424-overexpressing DU145-LN4 clones (424-2 and 424-8) shown. Upper right panel shows reduced E-Cadherin, EpCAM and γ-catenin and increased vimentin levels by Western blot. Bar chart of real time PCR data confirmed upregulation of miR-424 level in stable cell lines after normalizing to housekeeping small RNA, SNORD38B. (E) Transient transfection of premiR-424 in epithelial MCF-7 breast cancer cells resulted in increased cell scattering, relative to two individual control premiRNAs, as shown by phase contrast microscopy (scale bar 50 μm). Western blot showed decreased E-Cadherin and EpCAM expression in premiR-424-treated cells. β-actin shown as loading control. Each membrane was stripped and reprobed for each subsequent marker in (C), (D), and (E). Images were cropped to save space; original images are shown in Supplemental Figure 5.
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f6: MiRNA-424 regulates the epithelial phenotype.(A) Progressive downregulation of miR-424 expression in the DU145-LN cell series was measured by real time RT- PCR. (B) DU145-LN4 cells were transfected with two different control premiRs (left and right panels) or premiR-424 (center panel). PremiR-424 induced EMT-like changes, as observed by reduced cell-cell interactions in phase-contrast microscopy, and decreased E-cadherin and EpCAM levels in immunostaining. (C) Western blot analysis of DU145-LN4 cells treated with premiR controls, premiR-424 or mock transfected, collected at 6 and 21 days shows premiR-424 inhibited E-cadherin and EpCAM expression and increased vimentin levels. Actin shown as loading control. (D) Stable overexpression of miR-424 in DU145-LN4 cells resulted in loss of cell-cell contacts as shown by phase contrast microscopy (scale bar 100 μm). DU145-LN4 vector control (v3), and two miR-424-overexpressing DU145-LN4 clones (424-2 and 424-8) shown. Upper right panel shows reduced E-Cadherin, EpCAM and γ-catenin and increased vimentin levels by Western blot. Bar chart of real time PCR data confirmed upregulation of miR-424 level in stable cell lines after normalizing to housekeeping small RNA, SNORD38B. (E) Transient transfection of premiR-424 in epithelial MCF-7 breast cancer cells resulted in increased cell scattering, relative to two individual control premiRNAs, as shown by phase contrast microscopy (scale bar 50 μm). Western blot showed decreased E-Cadherin and EpCAM expression in premiR-424-treated cells. β-actin shown as loading control. Each membrane was stripped and reprobed for each subsequent marker in (C), (D), and (E). Images were cropped to save space; original images are shown in Supplemental Figure 5.

Mentions: We discovered a strong inverse correlation of miR-424 expression with the epithelial phenotype in DU145 cells. MiR-424 was the most significantly downregulated miRNA in our microarray analysis (Figure 4), and the level progressively decreased in the DU145-LN cell series as the cells became more epithelial. In microarray data DU145-LN1 showed 1.6 fold, DU145-LN2, 2.1 fold, and DU145-LN4 2.4 fold down-regulation of miR-424 expression, relative to parental DU145 cells (Figure 4). We confirmed this change using real time RT-PCR for miR-424 using RNA prepared from three individual cell cultures. Expression was normalized to the small housekeeping RNA, SNORD38B (Figure 6A). In real time RT-PCR, DU145-LN1 showed only 66% miR-424 expression, LN2 55%, LN3 26% and LN4 25% miR-424 expression, relative to parental DU145 cells (100%).


Regulation of epithelial plasticity by miR-424 and miR-200 in a new prostate cancer metastasis model.

Banyard J, Chung I, Wilson AM, Vetter G, Le Béchec A, Bielenberg DR, Zetter BR - Sci Rep (2013)

MiRNA-424 regulates the epithelial phenotype.(A) Progressive downregulation of miR-424 expression in the DU145-LN cell series was measured by real time RT- PCR. (B) DU145-LN4 cells were transfected with two different control premiRs (left and right panels) or premiR-424 (center panel). PremiR-424 induced EMT-like changes, as observed by reduced cell-cell interactions in phase-contrast microscopy, and decreased E-cadherin and EpCAM levels in immunostaining. (C) Western blot analysis of DU145-LN4 cells treated with premiR controls, premiR-424 or mock transfected, collected at 6 and 21 days shows premiR-424 inhibited E-cadherin and EpCAM expression and increased vimentin levels. Actin shown as loading control. (D) Stable overexpression of miR-424 in DU145-LN4 cells resulted in loss of cell-cell contacts as shown by phase contrast microscopy (scale bar 100 μm). DU145-LN4 vector control (v3), and two miR-424-overexpressing DU145-LN4 clones (424-2 and 424-8) shown. Upper right panel shows reduced E-Cadherin, EpCAM and γ-catenin and increased vimentin levels by Western blot. Bar chart of real time PCR data confirmed upregulation of miR-424 level in stable cell lines after normalizing to housekeeping small RNA, SNORD38B. (E) Transient transfection of premiR-424 in epithelial MCF-7 breast cancer cells resulted in increased cell scattering, relative to two individual control premiRNAs, as shown by phase contrast microscopy (scale bar 50 μm). Western blot showed decreased E-Cadherin and EpCAM expression in premiR-424-treated cells. β-actin shown as loading control. Each membrane was stripped and reprobed for each subsequent marker in (C), (D), and (E). Images were cropped to save space; original images are shown in Supplemental Figure 5.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3818652&req=5

f6: MiRNA-424 regulates the epithelial phenotype.(A) Progressive downregulation of miR-424 expression in the DU145-LN cell series was measured by real time RT- PCR. (B) DU145-LN4 cells were transfected with two different control premiRs (left and right panels) or premiR-424 (center panel). PremiR-424 induced EMT-like changes, as observed by reduced cell-cell interactions in phase-contrast microscopy, and decreased E-cadherin and EpCAM levels in immunostaining. (C) Western blot analysis of DU145-LN4 cells treated with premiR controls, premiR-424 or mock transfected, collected at 6 and 21 days shows premiR-424 inhibited E-cadherin and EpCAM expression and increased vimentin levels. Actin shown as loading control. (D) Stable overexpression of miR-424 in DU145-LN4 cells resulted in loss of cell-cell contacts as shown by phase contrast microscopy (scale bar 100 μm). DU145-LN4 vector control (v3), and two miR-424-overexpressing DU145-LN4 clones (424-2 and 424-8) shown. Upper right panel shows reduced E-Cadherin, EpCAM and γ-catenin and increased vimentin levels by Western blot. Bar chart of real time PCR data confirmed upregulation of miR-424 level in stable cell lines after normalizing to housekeeping small RNA, SNORD38B. (E) Transient transfection of premiR-424 in epithelial MCF-7 breast cancer cells resulted in increased cell scattering, relative to two individual control premiRNAs, as shown by phase contrast microscopy (scale bar 50 μm). Western blot showed decreased E-Cadherin and EpCAM expression in premiR-424-treated cells. β-actin shown as loading control. Each membrane was stripped and reprobed for each subsequent marker in (C), (D), and (E). Images were cropped to save space; original images are shown in Supplemental Figure 5.
Mentions: We discovered a strong inverse correlation of miR-424 expression with the epithelial phenotype in DU145 cells. MiR-424 was the most significantly downregulated miRNA in our microarray analysis (Figure 4), and the level progressively decreased in the DU145-LN cell series as the cells became more epithelial. In microarray data DU145-LN1 showed 1.6 fold, DU145-LN2, 2.1 fold, and DU145-LN4 2.4 fold down-regulation of miR-424 expression, relative to parental DU145 cells (Figure 4). We confirmed this change using real time RT-PCR for miR-424 using RNA prepared from three individual cell cultures. Expression was normalized to the small housekeeping RNA, SNORD38B (Figure 6A). In real time RT-PCR, DU145-LN1 showed only 66% miR-424 expression, LN2 55%, LN3 26% and LN4 25% miR-424 expression, relative to parental DU145 cells (100%).

Bottom Line: DU145-LN4 showed increased cell-cell adhesions, higher expression of multiple epithelial markers, such as E-cadherin, EpCAM and cytokeratin 18, and reduced expression of mesenchymal markers such as vimentin.Ectopic transient and stable miR-424 expression induced EMT, with reduced epithelial marker expression and increased cell scattering.We show that this cellular plasticity can be mediated through the combined action of miR-424 and the miR-200 family.

View Article: PubMed Central - PubMed

Affiliation: 1] Vascular Biology Program, Boston Children's Hospital, Karp Family Research Laboratories, 300 Longwood Ave., Boston, MA 02115 [2] Department of Surgery, Harvard Medical School, Boston, MA 02115.

ABSTRACT
Using an in vivo cycling strategy, we selected metastatic cancer cells from the lymph nodes (LN) of mice bearing orthotopic DU145 human prostate tumors. Repeated rounds of metastatic selection (LN1-LN4) progressively increased the epithelial phenotype, resulting in a new model of tumor cell mesenchymal-epithelial transition (MET). DU145-LN4 showed increased cell-cell adhesions, higher expression of multiple epithelial markers, such as E-cadherin, EpCAM and cytokeratin 18, and reduced expression of mesenchymal markers such as vimentin. The MET in DU145-LN4 cells was accompanied by increased expression of the miR-200 family, and antimiRs to miR-200c and miR-141 induced an EMT. MET also correlated with the loss of miR-424. Ectopic transient and stable miR-424 expression induced EMT, with reduced epithelial marker expression and increased cell scattering. Our model provides evidence for spontaneous MET in vivo. We show that this cellular plasticity can be mediated through the combined action of miR-424 and the miR-200 family.

Show MeSH
Related in: MedlinePlus