Downregulation of Pdcd4 by mir-21 facilitates glioblastoma proliferation in vivo

Abstract

MicroRNAs (miRNAs) are small, noncoding RNAs that play a critical role in developmental and physiological processes and are implicated in the pathogenesis of several human diseases, including cancer. They function by regulating target gene expression post-transcriptionally. In this study, we examined the role of oncogenic mir-21 in the pathogenesis of glioblastoma, the most aggressive form of primary brain tumor. We have previously reported that mir-21 is expressed at higher levels in primary glioblastoma-tissue and glioblastoma-derived cell lines than in normal brain tissue. We demonstrate that downregulation of mir-21 in glioblastoma-derived cell lines results in increased expression of its target, programmed cell death 4 (Pdcd4), a known tumor-suppressor gene. In addition, our data indicate that either downregulation of mir-21 or overexpression of its target, Pdcd4, in glioblastoma-derived cell lines leads to decreased proliferation, increased apoptosis, and decreased colony formation in soft agar. Using a glioblastoma xenograft model in immune-deficient nude mice, we observe that glioblastoma-derived cell lines in which mir-21 levels are downregulated or Pdcd4 is over-expressed exhibit decreased tumor formation and growth. Significantly, tumors grow when the glioblastoma-derived cell lines are transfected with anti-mir-21 and siRNA to Pdcd4, confirming that the tumor growth is specifically regulated by Pdcd4. These critical in vivo findings demonstrate an important functional linkage between mir-21 and Pdcd4 and further elucidate the molecular mechanisms by which the known high level of mir-21 expression in glioblastoma can attribute to tumorigenesis--namely, inhibition of Pdcd4 and its tumor-suppressive functions.

Fig. 1.

Mir-21 is highly expressed in glioblastoma (GBM) primary tumors and glioblastoma-derived cell lines compared to normal brain tissue. (A) Northern blot analysis of mir-21 in primary tissues (2H, 4A10, 35A28, and 52A28) and GBM-derived cell lines (SNB19, U251, U87, and SF767), compared with normal human brain samples (NB4330 and NB4126). Twenty micrograms of total RNA was loaded in each well. U6 loading controls from the same blot are shown in the lower panel. (B) Western blot analysis of mir-21 in primary tissues (2H, 4A10, 35A28, and 52A28) and GBM-derived cell lines (SNB19, U251, U87, and SF767), compared with normal human brain samples (NB4330 and NB4126). Forty micrograms of protein from whole-cell lysates were loaded in each well for quantifying Pdcd4 protein levels by Western blot analysis using an anti-Pdcd4 antibody. β-Actin loading controls from the same blots are shown in the lower panels. (C) Real-time polymerase chain reaction (PCR) quantification of mir-21 in primary GBM tissues, GBM-derived cell lines, and normal human brain samples. Stem-loop primers were used for reverse transcription followed by real-time PCR. mir-21 levels are shown as fold change versus the level detected in normal brain tissue. RNA input was normalized using 4 endogenous controls: 18S rRNA, β2M, GAPDH, and β-actin.

Fig. 2.

Inhibition of mir-21 expression in glioblastoma-derived cell lines enhances Pdcd4 expression. (A) Northern blot analysis of mir-21 expression levels in U251 and U87 cells following anti-mir-21 (30 μM) transfection. Total RNA was collected 24, 48, and 72 h after transfection, and 20 μg were loaded in each well for quantifying mir-21 levels by Northern blot analysis. U6 loading controls from the same blot are shown in the lower panel. (B) Western blot analysis of Pdcd4 in U251 and U87 cells after anti-mir-21 (30 μM) transfection. Whole-cell lysates were prepared 24, 48, and 72 h after transfection with anti-mir-21, and 40 μg of protein were loaded in each well for quantifying Pdcd4 protein levels by Western blot analysis using an anti-Pdcd4 antibody. β-Actin loading controls from the same blots are shown in the lower panels. In all blots, lane 1 is an untreated controls and lane 2 contains lysates from cells treated with a nonspecific negative/toxicity anti-mir (NS) control.

Fig. 3.

Anti-mir-21 treatment inhibits proliferation of glioblastoma-derived cell lines in vitro. (A) U251 and U87 cells that were untreated or transfected with a nonspecific negative/toxicity anti-mir (NS) control (30 μM) or anti-mir-21 (3, 10, and 30 μM) were examined. Fifty thousand cells were plated onto a 10-cm tissue culture dish at day 0 for each condition, and cells were counted daily. (B) Cell-cycle analyses of U251 and U87 cells and cells transfected with non-specific negative/toxicity anti-mir (NS) control (30 μM) or anti-mir-21 (30 μM) as well as U251^Pdcd4 and U87^Pdcd4 cells. Cells were fixed with 70% ethanol, treated with RNAse and stained with propidium iodide, after which cell-cycle progression was analyzed by flow cytometry. The data are shown as the percentage of cells in various phases of the cell cycle and are mean values ± standard errors of the mean of the percentage of G0/G1, G2/M, or S phase in 3 experiments. (C) TUNEL staining of U251 cells and U251 transfected with anti-mir-21 (30 μM, 72 h) or a nonspecific anti-mir (NS) control (30 μM) and U251^Pdcd4 cells. Untreated paraformaldehyde-fixed U251 cells treated with DNAse were included as a positive control. Cell nuclei were also stained with Hoechst dye. (D) Quantification of TUNEL-positive cells per 200 randomly selected cells on 4 different evaluations per condition.

Fig. 4.

Suppression of mir-21 inhibits anchorage-independent growth of glioblastoma-derived cell lines. (A) Quantification of colonies formed by (A) U251 and (B) U87 cells treated with anti-mir-21 (30 μM) for 72 h and plated out to grow on soft agar in 6-well plates at either 2.5 × 10⁴ cells/well or 5 × 10⁴ cells/well. Also depicted are U251 and U87 cells that were transfected with anti-mir-21 (30 μM) followed by transfection with siRNA to Pdcd4 (10 μM) for 72 h Untreated U251 or U87 cells, nonspecific negative/toxicity anti-mir (NS) control (30 μM) treated cells, and cells over-expressing Pdcd4 (U251^Pdcd4 and U87^Pdcd4) were also evaluated. Colony numbers are the averages of 6 determinations per condition ± SEM. (C) Western blot analyses to determine Pdcd4 expression levels in U251 and U87 transfected with anti-mir-21 or NS control (30 μM) only or with anti-mir-21 (30 μM) followed by siRNA to Pdcd4 (10 μM) treatment as well as U251^Pdcd4 and U87^Pdcd4 cell lines. β-Actin loading controls are shown in the lower panels.

Fig. 5.

Downregulation of mir-21 or overexpression of Pdcd4 results in reduction of glioblastoma xenograft growth that is reversed by siRNA to Pdcd4. (A) U251^GFP or U87^GFP (5 × 10⁶) cells that were either untreated, transfected with NS control (30 μM) or anti-mir-21 (30 μM), or transfected with anti-mir-21 (30 μM) followed by transfection with siRNA to Pdcd4 (10 μM) for 72 h, as well as cells expressing elevated levels of Pdcd4 (U251^GFP+Pdcd4 and U87^GFP+Pdcd4), were injected subcutaneously in the flanks of nude mice. The Xenogen Imaging System was used to image tumor growth in vivo. Data are representative of 5 independent xenograft experiments (total n = 24 animals per condition). (B) Northern blot analysis of mir-21 in U251^GFP and U87^GFP cells prior to their use as xenografts. U6 loading controls are shown in the lower panel. (C) Tumor volume of xenografts. Tumors were measured every 4 days from day 10 to day 34 after injection. Data are averages of 5 independent xenograft experiments (total n = 24 animals per condition) ± SEM. (D) Western blot analysis of Pdcd4 protein in tumors isolated from mice that were injected with U251^Pdcd4 or U87^Pdcd4 cell lines.