PMC

MiR-124 is downregulated and CDK6 is upregulated in medulloblastoma. (A) Expression of miR-124 in individual medulloblastoma tumor samples and medulloblastoma cell lines measured relative to U38 small nucleolar RNA and normalized to expression in fetal cerebellum. N1: adult cerebellum, N2: fetal cerebellum, T1–T8: medulloblastoma tumors, C1: D283 cell line, C2: D341 cell line, C3: D425 cell line. (B) Expression of CDK6 protein in control tissues, individual tumor samples, and medulloblastoma cell lines.

MiR-124 inhibits tumor growth in vivo. (A) Subcutaneous growth of D425 tumors expressing inducible miR-124 clone B is inhibited in animals provided doxycycline. (B) Tumors were visibly smaller at the time animals were sacrificed. (C) Ki67 staining of tumors demonstrated a marked reduction in the percentage of Ki67-positive cells (2 tumors from each group, 4 fields each; SE of the mean; P = .0006). Unpaired t-test was used to determine differences between the 2 groups.

Inducible expression of miR-124 in D425 medulloblastoma cells. (A) Doxycycline stimulation of lentiviral transduced D425 cells leads to increased expression of miR-124. Expression of miR-124 is measured relative to let-7a (white) and miR-16 (black). Two independently transduced D425–miR-124 cell lines (a and b) were tested. Expression differences following doxycycline stimulation were analyzed using Student's t-test; *P < .005. (B) Doxycycline stimulation leads to decreased levels of CDK6 in pTRIPZ–miR-124 lentiviral transduced D425 cells. (C) Cell numbers were determined at 96 h after doxycycline stimulation. Values represent mean ± SE (n ≥ 3) from replicate experiments (n = 2) normalized to the cell count in non-doxycycline-stimulated samples. Differences in cell numbers were analyzed using Student's t-test; *P < .05; **P < .0001.

MiR-124 inhibits proliferation of CDK6-expressing D425 medulloblastoma cells and induces G₀/G₁ cell cycle arrest miR-124. (A) Cell numbers were determined at 72 h after transfection of 100 nM miR-124 or negative control oligonucleotides to D283, D341, and D425 medulloblastoma cells. Values represent mean ± SD of replicate experiments normalized to the cell count in the control samples (D283 n = 51; D341 n = 34; D425 n = 14). Differences between miR-124 and control transfected cells were analyzed using Student's t-test; *P < .05. **P < 2e-7. ***P < 4e-15. (B) Cell cycle analysis conducted by fluorescence-activated cell sorter (FACS) at 48h after transfection of 100 nM miR-124 or negative control oligonucleotides to D425 cells. Cells were treated with BrdU for 30 min, fixed, treated with fluorescein isothiocyanate (FITC)–labeled anti-BrdU antibody and the DNA stain 7-amino-actinomycin D, and subjected to flow cytometry. Values represent mean ± SD (n = 2) of replicate experiments (n = 3). Differences between miR-124 and control transfected cells were analyzed using Student's t-test; *P < .0005; **P < 2e-6. (C) Cell cycle analysis was conducted by FACS at 48 h after transfection of 100 nM CDK6 siRNAs, miR-124, or negative control oligonucleotides to D425 cells. Cells were treated with BrdU for 30min, fixed, treated with FITC-labeled anti-BrdU antibody and the DNA stain 7-amino-actinomycin D, and subjected to flow cytometry. Values represent mean ± SD (n = 2) of replicate experiments (n = 3). Differences between the different transfections were analyzed using Student's t-test; *P < 3e-6. (D) Cell cycle analysis was conducted by FACS at 48 h after transfection of 100 nM CDK6 siRNAs, miR-124, or negative control oligonucleotides to D283 cells. Values represent mean ± SD (n = 2) of replicate experiments (n = 3). Differences between the different transfections were analyzed using Student's t-test; *P < .0001.