doi: 10.1002/stem.101.
miR-200 regulates PDGF-D-mediated epithelial-mesenchymal transition, adhesion, and invasion of prostate cancer cells
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- PMID:19544444
- PMCID: PMC3400149
- DOI: 10.1002/stem.101
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miR-200 regulates PDGF-D-mediated epithelial-mesenchymal transition, adhesion, and invasion of prostate cancer cells
Dejuan Kong et al. Stem Cells.2009 Aug.
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Abstract
MicroRNAs have been implicated in tumor progression. Recent studies have shown that the miR-200 family regulates epithelial-mesenchymal transition (EMT) by targeting zinc-finger E-box binding homeobox 1 (ZEB1) and ZEB2. Emerging evidence from our laboratory and others suggests that the processes of EMT can be triggered by various growth factors, such as transforming growth factor beta and platelet-derived growth factor-D (PDGF-D). Moreover, we recently reported that overexpression of PDGF-D in prostate cancer cells (PC3 PDGF-D cells) leads to the acquisition of the EMT phenotype, and this model offers an opportunity for investigating the molecular interplay between PDGF-D signaling and EMT. Here, we report, for the first time, significant downregulation of the miR-200 family in PC3 PDGF-D cells as well as in PC3 cells exposed to purified active PDGF-D protein, resulting in the upregulation of ZEB1, ZEB2, and Snail2 expression. Interestingly, re-expression of miR-200b in PC3 PDGF-D cells led to reversal of the EMT phenotype, which was associated with the downregulation of ZEB1, ZEB2, and Snail2 expression, and these results were consistent with greater expression levels of epithelial markers. Moreover, transfection of PC3 PDGF-D cells with miR-200b inhibited cell migration and invasion, with concomitant repression of cell adhesion to the culture surface and cell detachment. From these results, we conclude that PDGF-D-induced acquisition of the EMT phenotype in PC3 cells is, in part, a result of repression of miR-200 and that any novel strategy by which miR-200 could be upregulated would become a promising approach for the treatment of invasive prostate cancer.
Figures
Over-expression of PDGF-D induces EMT in PC3 cells. (A) Western blot analysis showed the expression of PDGF-D, transcription repressors and other mesenchymal as well as epithelial markers in PC3 Neo and PC3 PDGF-D cells, passage 15 and 20. (GAPDH protein was used as protein loading control). (B) Total cell lysates, cytosol and nuclear extracts were prepared from PC3 Neo and PC3 PDGF-D cells, passage 8, 15 and 20. The result from Western blot showed the expression of ZEB1, which mainly localized in the nuclear. GAPDH was used for protein loading control for cell lysates and cytosolic extracts, whereas retinoblastoma (RB) protein was used for protein loading control for nuclear extracts. (C) Photomicrographs of cells are shown: PC3 Neo cells display rounded epithelial cell shape (upper panel) and PC3 PDGF-D cells exhibit a fibroblastic-type phenotype (lower panel); original magnification, 200×. (N: PC3 Neo cells, D: PC3 PDGF-D cells, p8: passage 8).
Over-expression of PDGF-D in PC3 cells up-regulates the expression of transcription factors and down-regulates expressions of epithelial specific genes. (A) Real time RT-PCR was used to determine mRNA levels of epithelial markers in PC3 Neo and PC3 PDGF-D cells. Relative mRNA levels were normalized to GAPDH. (B) Real time RT-PCR was used to quantify the expression of ZEB1, ZEB2 and vimentin mRNA in PC3 Neo and PC3 PDGF-D cells. GAPDH was used for internal control to correct for the potential variation in RNA loading. (C) The results from real time RT-PCR showed the increased mRNA levels of E-cadherin, F11R, CRB3, and sciellin in PC3 PDGF-D transfected with ZEB1 siRNA compared to cells transfeced with control siRNA. Relative mRNA levels were normalized to GAPDH. (D) The decreased mRNA levels of ZEB1, ZEB2 and vimentin were observed in PC3 PDGF-D transfected with ZEB1 siRNA compared to control siRNA. Relative mRNA levels were normalized to GAPDH. (E) PC3 PDGF-D cells were transfected with ZEB1 or control siRNA and incubated for 72 h. Western blot analysis was performed using primary antibodies against ZEB1 and GAPDH. GAPDH was used for protein loading control. *, p< 0.05, **, p< 0.01 compared to control cells. (Neo: PC3 Neo cells, PDGF-D: PC3 PDGF-D cells, Con: control, E-cad: E-cadherin, Vim: vimentin).
miR-200 regulates the expressions of transcription factors and protein expressions associated with cell polarity, tight junctions, desmosomes, gap junctions and cell surface receptor. (A) Levels of miR-200a, miR-200b, miR-200c and miR-16 from PC3 Neo and PC3 PDGF-D cells were determined by using the miRNA-specific Taqman MGB probes and primers. The relative amount of miRNA was normalized to RNU6B. (B) The results from Western blot showed that expressions of ZEB1, ZEB2, snail2 and vimentin were significantly inhibited, while expression of E-cadherin was increased in PC3 PDGF-D cells transfected with miR-200b compared to PC3 PDGF-D cells transfected with control miRNA. GAPDH was used for protein loading control. (C) Real time RT-PCR was used to quantify the mRNA levels of ZEB1, ZEB2, snail2 and vimentin as well as mRNA levels of E-cadherin, stratifin, EpCAM, F11R and connexin 26 in PC3 PDGF-D cells transfected with miR-200b compared to transfection with negative control miRNA. Relative mRNA levels were normalized to GAPDH. (N: negative control miRNA, M: miR-200b, Neg: negative control miRNA, 200a, 200b and 200c: miR-200a, miR-200b, miR-200c, respectively).
MiR-200b reverses EMT phenotype of PC3 PDGF-D cells. (A) Photographs of cells are shown: PC3 PDGF-D cells transfected with negative control miRNA exhibit a fibroblastic-type phenotype (upper panel), PC3 PDGF-D cells transfected with miR-200b display round-like epithelial cell shape and cells form a cluster (lower panel). Original magnification, 200 ×. After 21 days of transfection, PC3 PDGF-D cells transfected with negative control miRNA or miR-200b were immunostained for the expressions of E-cadherin (B), ZO-1 (C), vimentin (D), or stained with Alexa Fluor 594 phalloidin for F-actin (E) with DAPI for DNA to show cell nucleus, as described under method section. Arrows indicate changes in the expression or location of epithelial and mesenchymal markers. Original magnification, 200 ×.
MiR-200b inhibits the migration and invasion, and reduces attachment and detachment of PC3 PDGF-D cells. (A) The effects of miR-200b on cell migration and invasion of PC3 PDGF-D cells were determined by using 24 well Transwell Permeable Supports with 8 μM pores. For the invasion assay, PC3 PDGF-D cells transfected with miR-200b or control miRNA were seeded into the Transwell inserts coated with growth factor reduced Matrigel. For the migration assay, PC3 PDGF-D cells transfected with miR-200b or control miRNA were seeded into the Transwell inserts not coated with Matrigel. After 24 h incubation, the cells were stained with 4 μg/ml Calcein AM in PBS at 37°C for 1 h. The fluorescence of the cells from lower sides of inserts was read in ULTRA Multifunctional Microplate Reader at excitation/emission wavelengths of 485/530 nm. Values of relative fluorescence are shown. The values represent the comparative amount of invaded or migrated cells. Panel (B) and (C) showed that over-expression of PDGF-D significantly increased cell attachment and detachment of PC3 cells, respectively. (D) Transfection of PC3 PDGF-D cells with miR-200b dramatically inhibited the attachment and detachment of PC3 PDGF-D cells after 15 days of transfection. n = 4. *, p< 0.05, **, p< 0.01 compared to control cells.
Purified active PDGF-D treatment down-regulates the expression of miR-200 family and induces EMT. (A) The total RNA was isolated using Trizol reagent. miRNA levels were determined by using the miRNA-specific Taqman MGB probes and primers. The relative amount of miRNA was normalized to RNU6B. (B) Real time RT-PCR was used to quantify the expressions of ZEB1, ZEB2, snail2, E-cadherin and vimentin mRNA in PC3 cells treated with purified active PDGF-D protein for 4 weeks compared to PC3 cells treated with control. Relative mRNA levels were normalized to GAPDH. Panel (C) and (D) PC3 cells were treated with purified active PDGF-D for 10 or 20 days and then cell lysates were prepared. Western blot analysis showed that the expression of ZEB2 was increased and the expression of E-cadherin was decreased in a dose-dependent manner. (E) The effects of purified active PDGF-D treatment on cell detachment of PC3 cells were determined and results showed that cell detachment significantly increased in PC3 cells treated with PDGF-D for 4 weeks. (F) The effects of purified active PDGF-D treatment on migration of PC3 cells were determined by using 24 well Transwell Permeable Supports with 8 μM pores. Values of relative fluorescence are shown. The values represent the comparative amount of migrated cells. *, p< 0.05, **, p< 0.01 compared to control.
Over-expression of PDGF-D in LNCaP cells induces EMT characteristics. (A) Western blot analysis showed the expressions of ZO-1, vimentin, fibronectin and the levels of PDGF-D in LNCaP PDGF-D cells (PDGF-D over-expressing cells) compared to LNCaP Neo cells (GAPDH protein was used as protein loading control). (B) Levels of miR-200a, miR-200b, and miR-200c in LNCaP PDGF-D cells (PDGF-D) and LNCaP Neo cells (Neo) were determined by using the miRNA-specific Taqman MGB probes and primers. The relative amount of miRNA was normalized to RNU6B. (C) Real time RT-PCR was used to determine mRNA levels of PDGF-D in PC3 and LNCaP cells. Relative mRNA levels were normalized to GAPDH. (D) Levels of miR-200a, miR-200b and miR-200c from PC3 and LNCaP cells were determined by using the miRNA-specific Taqman MGB probes and primers. The relative amount of miRNA was normalized to RNU6B. (E) For the invasion assay, LNCaP cells transfected with anti-miR-200a, miR-200b and miR-200c (inhibitors) or anti-miRNA control were seeded into the Transwell inserts coated with growth factor reduced Matrigel. After 24 h incubation, the cells were stained with 4 μg/ml Calcein AM in PBS at 37°C for 1 h. The fluorescence of the cells at the bottom sides of the inserts was read in ULTRA Multifunctional Microplate Reader at excitation/emission wavelengths of 485/530 nm. Values of relative fluorescence are shown. The values represent the comparative values of invaded cells. [*, p< 0.05, **, p< 0.01 compared to control. N: LNCaP Neo cells, D: LNCaP PDGF-D cells, con: anti-miRNA control, 200abc: ant-miR-200a, miR-200b and miR-200c (miRNA inhibitors)].
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