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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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The miR-200 family regulates EMT in metastatic DU145-LN4 cells.(A) Real time RT-PCR confirmed increased expression of the miR-200 family members; miR-200a, b, c and miR-141 in DU145-LN2 and DU145-LN4 cells, relative to DU145 cells. Mean +/− SEM of triplicates. (B) Treatment of DU145-LN4 cells with a combination of antimiRs to the miR-200a + miR-200b (left center panel) or miR-200c + miR-141 (right center panel) reduced cell clustering and cell-cell interactions, as observed by phase-contrast microscopy (upper panels, scale bar = 100 μm), and E-cadherin immunostaining (lower panels, scale bar = 25 μm) at 14 days. Cells were transfected at day 0 and day 7. (C) Western blot at 7 days (following day 0 transfection) and (D) 14 days (following day 0 and day 7 transfection) showed reduced expression of E-cadherin and EpCAM, and increased vimentin expression following treatment of DU145-LN4 cells with antimiRs 200c + 141. To account for increased antimiR concentration when treated in combination, controls were treated with 200 nM (2X) and 400 nM (4X) non-specific antimiR. Actin shown as protein loading control. The same membrane was stripped and reprobed for each subsequent marker in C or D. Images were cropped to save space; original images are shown in Supplemental Figure 5. (E) Real time RT-PCR amplification of ZEB1, ZEB2 and CDH1 in DU145LN4 cells treated with antimiR-200c + 141 (grey bars), relative to antimiR control (black bars). Data normalized to GAPDH, mean +/− SD.
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f5: The miR-200 family regulates EMT in metastatic DU145-LN4 cells.(A) Real time RT-PCR confirmed increased expression of the miR-200 family members; miR-200a, b, c and miR-141 in DU145-LN2 and DU145-LN4 cells, relative to DU145 cells. Mean +/− SEM of triplicates. (B) Treatment of DU145-LN4 cells with a combination of antimiRs to the miR-200a + miR-200b (left center panel) or miR-200c + miR-141 (right center panel) reduced cell clustering and cell-cell interactions, as observed by phase-contrast microscopy (upper panels, scale bar = 100 μm), and E-cadherin immunostaining (lower panels, scale bar = 25 μm) at 14 days. Cells were transfected at day 0 and day 7. (C) Western blot at 7 days (following day 0 transfection) and (D) 14 days (following day 0 and day 7 transfection) showed reduced expression of E-cadherin and EpCAM, and increased vimentin expression following treatment of DU145-LN4 cells with antimiRs 200c + 141. To account for increased antimiR concentration when treated in combination, controls were treated with 200 nM (2X) and 400 nM (4X) non-specific antimiR. Actin shown as protein loading control. The same membrane was stripped and reprobed for each subsequent marker in C or D. Images were cropped to save space; original images are shown in Supplemental Figure 5. (E) Real time RT-PCR amplification of ZEB1, ZEB2 and CDH1 in DU145LN4 cells treated with antimiR-200c + 141 (grey bars), relative to antimiR control (black bars). Data normalized to GAPDH, mean +/− SD.

Mentions: MiR-200 family expression was investigated using real time RT-PCR. MiR-200a, miR-200b, miR-200c and miR-141 were amplified from three independent RNA collections of the parental DU145, DU145-LN2 and DU145-LN4 cells. Data were normalized to the small housekeeping RNA, SNORD38B. Figure 5A demonstrates that miR-200a, miR-200b, miR-200c and miR-141 expression were progressively increased in DU145-LN2 and DU145-LN4 cells, compared to DU145. MiR-200c and miR-141 showed a greater fold increase in DU145-LN4 relative to DU145 (>7X), compared to miR-200a or miR-200b (approximately 3X). These expression patterns correlated with the miR-200 family microarray data.


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)

The miR-200 family regulates EMT in metastatic DU145-LN4 cells.(A) Real time RT-PCR confirmed increased expression of the miR-200 family members; miR-200a, b, c and miR-141 in DU145-LN2 and DU145-LN4 cells, relative to DU145 cells. Mean +/− SEM of triplicates. (B) Treatment of DU145-LN4 cells with a combination of antimiRs to the miR-200a + miR-200b (left center panel) or miR-200c + miR-141 (right center panel) reduced cell clustering and cell-cell interactions, as observed by phase-contrast microscopy (upper panels, scale bar = 100 μm), and E-cadherin immunostaining (lower panels, scale bar = 25 μm) at 14 days. Cells were transfected at day 0 and day 7. (C) Western blot at 7 days (following day 0 transfection) and (D) 14 days (following day 0 and day 7 transfection) showed reduced expression of E-cadherin and EpCAM, and increased vimentin expression following treatment of DU145-LN4 cells with antimiRs 200c + 141. To account for increased antimiR concentration when treated in combination, controls were treated with 200 nM (2X) and 400 nM (4X) non-specific antimiR. Actin shown as protein loading control. The same membrane was stripped and reprobed for each subsequent marker in C or D. Images were cropped to save space; original images are shown in Supplemental Figure 5. (E) Real time RT-PCR amplification of ZEB1, ZEB2 and CDH1 in DU145LN4 cells treated with antimiR-200c + 141 (grey bars), relative to antimiR control (black bars). Data normalized to GAPDH, mean +/− SD.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f5: The miR-200 family regulates EMT in metastatic DU145-LN4 cells.(A) Real time RT-PCR confirmed increased expression of the miR-200 family members; miR-200a, b, c and miR-141 in DU145-LN2 and DU145-LN4 cells, relative to DU145 cells. Mean +/− SEM of triplicates. (B) Treatment of DU145-LN4 cells with a combination of antimiRs to the miR-200a + miR-200b (left center panel) or miR-200c + miR-141 (right center panel) reduced cell clustering and cell-cell interactions, as observed by phase-contrast microscopy (upper panels, scale bar = 100 μm), and E-cadherin immunostaining (lower panels, scale bar = 25 μm) at 14 days. Cells were transfected at day 0 and day 7. (C) Western blot at 7 days (following day 0 transfection) and (D) 14 days (following day 0 and day 7 transfection) showed reduced expression of E-cadherin and EpCAM, and increased vimentin expression following treatment of DU145-LN4 cells with antimiRs 200c + 141. To account for increased antimiR concentration when treated in combination, controls were treated with 200 nM (2X) and 400 nM (4X) non-specific antimiR. Actin shown as protein loading control. The same membrane was stripped and reprobed for each subsequent marker in C or D. Images were cropped to save space; original images are shown in Supplemental Figure 5. (E) Real time RT-PCR amplification of ZEB1, ZEB2 and CDH1 in DU145LN4 cells treated with antimiR-200c + 141 (grey bars), relative to antimiR control (black bars). Data normalized to GAPDH, mean +/− SD.
Mentions: MiR-200 family expression was investigated using real time RT-PCR. MiR-200a, miR-200b, miR-200c and miR-141 were amplified from three independent RNA collections of the parental DU145, DU145-LN2 and DU145-LN4 cells. Data were normalized to the small housekeeping RNA, SNORD38B. Figure 5A demonstrates that miR-200a, miR-200b, miR-200c and miR-141 expression were progressively increased in DU145-LN2 and DU145-LN4 cells, compared to DU145. MiR-200c and miR-141 showed a greater fold increase in DU145-LN4 relative to DU145 (>7X), compared to miR-200a or miR-200b (approximately 3X). These expression patterns correlated with the miR-200 family microarray data.

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