Article Figures & Data
Figures
- Figure 1.
Screening for miR-21 target genes that promote prostate cancer growth and androgen independence. A, 3′UTR reporter analysis of reported miR-21 target genes. Top, Alignment of reported miR-21 target genes and miR-21–binding sites. Numbers indicate nucleotide site within mRNA. Bottom, HEK 293 cells were transfected with the 3′UTR report vector for each gene, miR-21 or control mimic, and reference Renilla luciferase. Luciferase activity is normalized to Renilla and relative to miR-control. Bars represent mean + SE of at least five independent quadruplicate experiments. B, Androgen-dependent cell growth in CM as measured by MLuc Viability Assay, 7 days after miR-21 or siRNA transfection. C, Androgen-independent cell growth in ADM as measured by MLuc Viability Assay, 7 days after miR-21 or siRNA transfection. Growth is relative to control miRNA or control siRNA for each gene. Bars represent mean + SE of two independent triplicate experiments. *, P < 0.05.
- Figure 2.
Androgen signaling suppresses PDCD4 expression through miR-21. A, LNCaP and LAPC4 cells were transfected with 20 nmol/L of miR-21 mimic or inhibitor for 48 hours and PDCD4 expression quantified by Western blot analysis, normalized to ACTB. B, Cells were grown in ADM for 24 hours and then stimulated with indicated amounts of androgen (R1881) or CM for 24 additional hours (left); or cells were grown in CM condition for 24 hours and then incubated with indicated concentration of R1881 or ADM for 24 hours (right). PDCD4 expression quantified by Western blot analysis, normalized to ACTB. C, Cells were incubated in CM for 24 hours, then treated for 24 hours with bicalutamide or vehicle control. PDCD4 expression quantified by Western blot analysis, normalized to ACTB. D, Cells were transfected with 20 nmol/L of miR-21 inhibitor or control. The next day cells were exposed to ADM, or ADM supplemented with 10 nmol/L R1881, for 24 hours. PDCD4 expression quantified by Western blot analysis, normalized to GAPDH. FC, fold change relative to control. E, Schematic of suppressed PDCD4 expression through androgen signaling and miR-21.
- Figure 3.
PDCD4 and miR-21 expression are inversely correlated and associated with aggressive prostate cancer. Analysis of miR-21 miRNA, PDCD4 mRNA, and PDCD4 protein expression from the TCGA-PRAD. Spearman correlation analysis of normalized of miR-21 (RPM) and PDCD4 protein (RPPA) expression in 350 matching TCGA-PRAD samples (A), miR-21 (RPM) and PDCD4 mRNA (RPM) expression in 546 matching TCGA-PRAD samples (B), and PDCD4 mRNA and protein in 351 matching TCGA-PRAD samples (C). Association of PDCD4 protein (D), PDCD4 mRNA (E), and miR-21 with radical prostatectomy Gleason score in TCGA-PRAD tissue samples (F; one-way ANOVA).
- Figure 4.
PDCD4 knockdown induces androgen-dependent and androgen-independent prostate cancer growth. A and B, PDCD4 knockdown induced androgen-dependent and androgen-independent cell growth. LNCaP or LAPC4 cells were transiently transfected with 20 nmol/L of miR-control or miR-21 mimic, or 20 nmol/L of si-control or PDCD4 siRNAs. Forty-eight hours after transfection, cells were grown in CM, CM + 10 μmol/L bicalutamide, or ADM for 7 days. Cell viability was evaluated by MTS assay and reported relative to miR-control signal in CM. Mean + SE from 12 independent measurements. *, P < 0.05, relative to miR-control or si-control. C and D, Cells were cotransfected with miR-control or miR-21 mimic and pCMV6-empty vector (EV) or pCMV6-PDCD4. After 48 hours, cells were grown in CM, CM + 10 μmol/L bicalutamide, or ADM for 7 days. Cell viability was evaluated by MTS assay and reported relative to miR-control signal in CM. Mean + SE from 12 independent measurements (*, P < 0.05).
- Figure 5.
PDCD4 knockdown induces cell-cycle progression and inhibits apoptosis in a cell-specific manner. A, Stable PDCD4 knockdown lines. LNCaP or LAPC4-shControl and -shPDCD4 cells were generated by lentiviral transduction. PDCD4 protein expression measured by Western blot analysis, normalized to GAPDH. FC, fold change relative to shControl. B, LNCaP (left) or LAPC4 (right) cell growth in CM and ADM as measured by MTS assay (7 days). Mean + SE from 12 independent measurements. *, P < 0.05, relative to shControl. C, Cell-cycle analysis in CM or ADM by flow cytometry, 72 hours. Left figure represents one of three independent replicates and right graph shows combined three biologic replicates. Mean + SE (n = 3). *, P < 0.05. D, Apoptotic analysis by Annexin V staining and flow cytometry after 72 hours growth in CM or ADM. Left figure represents one of three independent replicates and right graph indicates total percentage of Annexin V+ cell population.
- Figure 6.
PDCD4 knockdown enhances tumorigenesis, increases tumor growth rate, and imparts castration resistance in vivo. A, Subcutaneous xenograft growth of LNCaP-shControl or LNCaP-shPDCD4 over time, initiating from the time of injection. Dashed line indicates castration when tumors reached approximately 350 mm3 volume. B, Tumor growth over time, normalized to the time of castration. Mean ± SE (LNCaP-shControl, n = 6; LNCaP-shPDCD4, n = 9). *, P < 0.05; two-Way ANOVA. C, Subcutaneous xenograft growth of LAPC4-shControl or LAPC4-shPDCD4 over time, initiating with the time of injection. Dashed line indicates castration when tumors reached approximately 380 mm3 volume. D, Tumor growth over time, normalized to the time of castration. Mean ± SE (LAPC4-shControl, n = 5; LAPC4-shPDCD4, n = 7; *, P < 0.05; two-Way ANOVA).
Additional Files
Supplementary Data
- Supplementary Tables S1-S2, Supplementary Figures S1-S5 - Supplementary Table S1: siRNA and shRNA Vectors Supplementary Table S2: The sequence of primers used in this study Supplementary Figure S1. miR-21 suppresses PDCD4 expression in normal prostate epithelial cells. Supplementary Figure S2. Androgen signaling and miR-21 suppresses PDCD4 mRNA expression. Supplementary Figure S3. Transient PDCD4 over-expression and PDCD4 knockdown. Supplementary Figure S4. Transient PDCD4-overexpression induces apoptosis of LAPC4, but not LNCaP, cells. Supplementary Figure S5. PDCD4 expression regulates cell proliferation and apoptosis.
Volume 17, Issue 2
