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Molecular Cancer Therapeutics
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MicroRNA-200c mitigates invasiveness and restores sensitivity to microtubule-targeting chemotherapeutic agents

Dawn R. Cochrane, Nicole S. Spoelstra, Erin N. Howe, Steven K. Nordeen and Jennifer K. Richer
Dawn R. Cochrane
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Nicole S. Spoelstra
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Erin N. Howe
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Steven K. Nordeen
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Jennifer K. Richer
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DOI: 10.1158/1535-7163.MCT-08-1046 Published May 2009
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    Figure 1.

    MiR-200c and ZEB1 are inversely correlated in endometrial, breast, and ovarian cancer cells. A, RNA and protein were harvested from endometrial cancer cell lines—AN3CA and Hec50 (high-grade, representing type 2 endometrial cancers), and Ishikawa (representing type 1 endometrial cancer), EEC B37 (hTERT transformed normal endometrial epithelial cells), and HIESC (SV40 transformed normal endometrial stromal cells). RNA was assayed for miR-200c by real-time PCR (top). Immunoblots of whole-cell protein extracts were probed for ZEB1, E-cadherin, N-cadherin, vimentin, and α-tubulin as a loading control (bottom). B, RNA and protein were harvested from aggressive breast cancer cell lines (BT-549 and MDA 231) as well as the more differentiated cell lines (BT-474, MCF7, T47D, and ZR75) for detection of miR-200c and immunoblot analysis of epithelial and mesenchymal markers. C, ovarian cell lines (2008, Hey, SKOV3, OVCA 420, and OVCA 433) were harvested and assayed as above. Each graph is representative of three independent experiments. For real-time RT-PCR, each column represents the mean of quadruplicate samples and bars represent SE. MiR-200c levels are normalized to rRNA and are relative to AN3CA, MDA-MB-231, or 2008 cells, respectively.

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    Figure 2.

    Addition of exogenous miR-200c results in repression of ZEB1 and restoration of E-cadherin protein. Hec50 endometrial cancer cells (A) and MDA-MB-231 breast cancer cells (B) were treated with transfection reagent only (mock), scrambled negative control mimic (negative), or miR-200c mimic (pre-200c). After 48 h, RNA was harvested and miR-200c levels were determined by real-time PCR (top). Columns, mean of quadruplicate samples; bars, SE. The miR-200c levels are normalized to rRNA and are relative to mock transfection levels. Western blots of protein from the three experimental groups (mock, negative, or miR-200c treated) were probed for ZEB1, E-cadherin, and α-tubulin as a loading control. Three replicates per treatment group are shown. For both real-time RT-PCR and Western blots, results are representative of one of three independent experiments. C, Hec50 cells grown on coverslips were treated as above and fluorescent immunocytochemistry results using antibodies recognizing ZEB1 (red), E-cadherin (green), and 4′,6-diamidino-2-phenylindole (DAPI; blue) are shown merged. Bottom, relevant IgG-negative controls. Magnification, ×1,000.

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    Figure 3.

    Restoration of miR-200c expression in Hec50 decreases migration and invasion. A, Hec50 cells were treated with transfection reagent only, a scramble negative control, or miR-200c mimic. After 48 h, wounds were inflicted and pictures taken at 0, 4, 8, 12, and 24 h after wounding. Lines indicate width of the wound at time zero. Pictures shown are from one experiment representative of three separate experiments (not shown). B, mock-, negative-, or miR-200c–transfected cells were subjected to a transwell migration assay. After 48 h, cells on the bottom side of the membrane were stained and mounted onto slides and the mean number of cells in four fields of vision on a cross-hatch was counted with error bars representing SE of four replicates. *, statistically significant difference between the numbers of cells migrating in the pre-200c, compared with either mock-transfected cells or negative control–treated cells [P = 2.8 × 10−4 and P = 6.0 × 10−8, respectively (Student's t test)]. Representative images (×100 magnification) of stained filters are shown. C, the number of cells able to invade through Matrigel-coated Boyden chambers was also determined for each group. Columns, mean number of cells from four replicates; bars, SE. *, statistically significant difference between the pre-200c–treated group, compared with either mock-transfected cells or negative control–treated cells [P = 0.0039 and P = 0.0020, respectively (Student's t test)].

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    Figure 4.

    MiR-200c alters cell death in response to microtubule-targeting chemotherapeutic agents specifically. Hec50 cells were treated with transfection reagent only (mock), scrambled negative control (negative), or miR-200c mimic (pre-200c). Twenty-four hours after transfection, cells were treated with 0, 5, 10, 15, 20, or 25 nmol/L of paclitaxel (A) or with 0, 20, 30, 40, or 50 μmol/L of cisplatin (B) and, 24 h after drug treatment apoptosis, were assayed using a Cell Death ELISA. Points, percent maximum apoptosis; bars, SE of triplicate samples. This experiment was done twice and representative experiments for each drug are shown. *, P < 0.05, between pre-200c–treated cells and mock or negative controls (Student's t test). Hec50 cells treated with pre-200c or negative controls were treated with agents that cause apoptosis via cell surface receptors (TRAIL, 50 ng/mL or FasL, 125 ng/mL; C), DNA damage (doxorubicin, 1 μg/mL or mitomycin C, 6 μg/mL; D), or microtubule poisons (vincristine, 100 nmol/L or epothilone B, 100 nmol/L; E), and Cell Death ELISAs were done. Columns, mean of five replicates; bars, SE. The experiment was repeated on three separate occasions with the same result, and a representative experiment is shown. *, P < 0.05, between pre-200c and both the mock and negative controls (Student's t test).

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    Figure 5.

    Heatmap of genes significantly affected by restoration of miR-200c levels in Hec50 cells as determined by expression profiling. Hec50 cells were treated in triplicate with mock, negative control, or pre-200c transfection, and gene expression analysis was done on Affymetrix HGU133 Plus 2.0 oligonucleotide cDNA expression array chips. A, genes with a statistically significant (ANOVA) ≥1.5-fold up-regulation (red) or down-regulation (green) in the pre-200c–treated cells versus both the negative control and the mock-transfected cells are shown in a heatmap. Expected alterations in E-cadherin (CDH1) and ZEB1 are highlighted. B, genes ≥1.5-fold up-regulated or down-regulated in the pre-200c–treated cells versus either negative control are shown in a separate heatmap. C, a heatmap of genes from A or B that are bioinformatically predicted to be targets of miR-200c. D, genes differentially regulated by miR-200c implicated in cell migration as determined by Ingenuity Pathway Analysis. Note that each gene was normalized to its average expression over the nine chips, such that the intensities center around 1 and are presented on a scale of −2 to +2 and are thus not indicative of relative fold changes. Fold changes and P values for these genes are listed in Supplementary Table S1.1

  • Figure 6.
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    Figure 6.

    Select genes altered by restoration of miR-200c are validated by RT-PCR. SYBR Green real-time RT-PCR was done on Hec50 cells treated with transfection reagent only (mock), 60 nmol/L of a scramble negative control (negative), or 60 nmol/L of the miR-200c mimic (pre-200c) using primers specific for CHK2, ARHGDIB, EPHB1, MAL2, LEPR, and ST6GALNAC5. Bars, SE of three replicates.

  • Figure 7.
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    Figure 7.

    A site in the TUBB3 3′-UTR is a direct target of miR-200c, and a decrease in TUBB3 protein corresponds with an increase in cell death in response to microtubule-targeting agents. A, SYBR Green real-time RT-PCR using primers specific for TUBB3 was done on RNA from Hec50 cells treated with miR-200c mimic (pre-200c), negative scrambled control (negative), or mock-transfected (mock) control (top left). A corresponding Western blot consisting of protein from the same cells was probed for TUBB3 (bottom left) and PSTAIR (used as a loading control). A fragment of the TUBB3 3′-UTR (located 117–379 bp after the stop codon) containing the putative miR-200c binding site, or the same fragment with the indicated base pairs that bind to the miR-200c seed sequence mutated, was cloned into the luciferase reporter vector pMIR-REPORT. These constructs, empty pMIR-REPORT vector, vector containing wild-type TUBB3 3′-UTR, or mutated TUBB3 3′-UTR (TUBB3 UTR mut), were transfected into Hec50 cells following negative control, pre-200c, or mock transfection. A dual reporter luciferase assay was done, and relative luciferase units (RLU) were calculated as firefly luciferase values divided by renilla values. Columns, mean of five replicate samples; bars, SE. *, P = 0.041, statistically significant difference in the amount of luciferase detected when the wild-type TUBB3 UTR is in the presence of pre-200c versus negative control. **, P = 0.006, difference in the amount of luciferase in the presence of pre-200c and either the empty vector or wild-type TUBB3 3′-UTR–containing reporter. ***, P = 0.003, difference between the amounts of luciferase measured when it is targeted by wild-type versus mutated TUBB3 3′-UTR. B, an aggressive ovarian cancer cell line, Hey, was treated with pre-200c, a negative control, or mock transfection. Top left, Western blot for TUBB3 with PSTAIR as a loading control. A Cell Death ELISA was done on the mock-, negative control–, or pre-200c–transfected Hey cells treated with various chemotherapeutic agents. Top right, Trail and FasL; bottom left, cisplatin, doxorubicin, and mitomycin C; bottom right, paclitaxel, vincristine, and epothilone B. Asterisks, statistically significant differences (as determined by Student's t tests) between the pre-200c–treated cells versus negative- and mock-transfected controls individually (P = 5.8 × 10−5 and P = 7.9 × 10−5, pre-200c versus mock and negative control, respectively, in the paclitaxel-treated group; P = 0.0005 and P = 0.0009, pre-200c versus mock and negative control, respectively, in the vincristine-treated group; and P = 1.3 × 10−5 and P = 2.3 × 10−5, pre-200c versus mock and negative control, respectively, in the epothilone B–treated group).

Additional Files

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    • Supplementary Table S1
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Molecular Cancer Therapeutics: 8 (5)
May 2009
Volume 8, Issue 5
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MicroRNA-200c mitigates invasiveness and restores sensitivity to microtubule-targeting chemotherapeutic agents
Dawn R. Cochrane, Nicole S. Spoelstra, Erin N. Howe, Steven K. Nordeen and Jennifer K. Richer
Mol Cancer Ther May 1 2009 (8) (5) 1055-1066; DOI: 10.1158/1535-7163.MCT-08-1046

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MicroRNA-200c mitigates invasiveness and restores sensitivity to microtubule-targeting chemotherapeutic agents
Dawn R. Cochrane, Nicole S. Spoelstra, Erin N. Howe, Steven K. Nordeen and Jennifer K. Richer
Mol Cancer Ther May 1 2009 (8) (5) 1055-1066; DOI: 10.1158/1535-7163.MCT-08-1046
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