Fig 1.
Establishment of YB-1-silenced neuroblastoma cell strain.
(A) Expression levels of YB-1 were examined by Western blot analysis in several human neuroblastoma cell lines with β-actin as the internal control. (B-D) SH-SY5Y cells were transfected with YB-1 shRNA construct (SH-shYB-1) or non-targeting control construct (SH-shCON), then selected for stable expression. Cells derived from the positive monoclones were assessed for (B) YB-1 mRNA expression level by real-time PCR, (C) YB-1 protein distribution by immunofluorescence staining and (D) total YB-1 protein level by Western blot analysis. The figure shows the representative images of three independent experiments, and the values are expressed as mean ± standard deviation. Compared with SH-SY5Y control, ***P<0.001.
Fig 2.
YB-1 silencing suppressed proliferation and induced cell cycle arrest of SH-SY5Y cells.
(A) Proliferation of YB-1-silenced SH-SY5Y cells, namely SH-shYB-1, was assessed by MTT assay, in parallel with SH-SY5Y and SH-shCON as control. (B, C) The cells were fixed with 70% ethanol, stained with propidium iodide (PI), and analyzed by flow cytometry for cell cycle stages. B shows the statistical results of cell cycle analysis excluding the apoptotic cells (sub-G0/G1 peak) and C presents a set of representative FACS data of three independent experiments. Values are expressed as mean ± standard deviation. Compared with SH-SY5Y control, **P<0.01; ***P<0.001.
Fig 3.
YB-1 regulated Cyclin D1 transcription in neuroblastoma SH-SY5Y cells.
(A) Expression levels of cell cycle regulators such as Cyclin A and Cyclin D1 in SH-SY5Y, SH-shCON and SH-shYB-1 cells were examined by Western blot analysis. (B) A schematic illustration of the promoter region of CCND1 which encodes Cyclin D1 indicates the transcription start site (TSS), putative YB-1 binding site and the location of two sets of primers (CCND1p #1 and #2) used for chromatin-immunoprecipitation (ChIP) assay. (C) ChIP assay followed by PCR analysis was performed in SH-SY5Y cells to detect binding of YB-1 on the promoter region of CCND1. Binding of RNA polymerase II (RNA PolII) on GAPDH promoter, which was detected with primers for GAPDH promoter (GAPDHp) by PCR, was used as a positive control for ChIP experiments, whereas IgG served as a negative control for non-specific binding. (D) pGL-3 Firefly luciferase reporter plasmid containing a CCND1 promoter fragment (pGL-3-CCND1) or pGL-3 vector alone was transfected in combination with Renilla luciferase reporter vector pRL-TK into SH-SY5Y, SH-shCON and SH-shYB-1 cells. Luciferase activity representing activity of the promoter was quantified as the ratio of FL/RL which was then normalized to SH-SY5Y cells. Values are expressed as mean ± standard deviation. Compared with SH-SY5Y control, ***P<0.001.
Fig 4.
YB-1 silencing induced apoptosis in neuroblastoma cells.
(A) SH-SY5Y, SH-shCON and SH-shYB-1 cells were double stained with anti-Annexin-V-FITC antibody and PI, followed by FACS analysis for cell apoptosis. Cells that fell in the lower right quadrant were characterized as early apoptotic cells which were statistically analyzed in B. (C) Cells were incubated with Hoechst stain, whereby the fluorescent stain that was taken by the apoptotic cells bound to DNA and emitted fluorescence under a fluorescent microscope. (D) The levels of several critical apoptosis markers were examined by Western blot analysis with β-actin as the internal control. The figure shows the representative images of three independent experiments, and the values are expressed as mean ± standard deviation. Compared with SH-SY5Y control, ***P<0.001.
Fig 5.
YB-1-silenced SH-SY5Y cells exhibited reduced tumorigenicity and delayed tumor formation in vivo.
(A) SH-SY5Y, SH-shCON and SH-shYB-1 cells were seeded sparsely on culture dishes and allowed to form colonies for 2 weeks. Colonies consisting of more than 50 cells were counted and colony forming ratio was calculated. (B) 106 SH-SY5Y, SH-shYB-1 or SH-shCON cells were inoculated subcutaneously into an 8-week-old BALB/c nude mouse (n = 6 each group), and tumor volumes were measured externally with a caliper every week and calculated as previously described. (C, D) By end of week 8, the mice were sacrificed, and the tumors were excised, photographed and weighed. (E) Tumor tissues were fixed, paraffin embedded, sectioned and stained with hematoxylin and eosin (H&E) for histological examination. (F) Tumor sections were permeabilized, enzyme inactivated, and incubated sequentially with TUNEL reaction solution, Converter-POD and DAB, followed by counterstaining with hematoxylin. Fragmented DNA was labeled and turned brownish under an optical microscope. (G) Tumor tissues were lysed and total proteins were extracted. Expression levels of YB-1 and Cyclin-D1 in the tumor cells were examined by Western blot analysis. E-G show representative images from all the experimental mice. Values are expressed as mean ± standard deviation. Compared with SH-SY5Y control, *P<0.05; ***P<0.001.
Fig 6.
Targeting YB-1 by intra-tumor shRNA injection inhibited tumor growth in mice.
SH-SY5Y cells were inoculated subcutaneously to 8-week-old mice at 106 per mouse. When the tumors grew to approximately 100 mm3, the mice were randomly assigned to receive an intra-tumor injection of PBS, shCON plasmids or shYB-1 plasmids (n = 6 each group). (A) Tumor volumes were measured every week for 8 weeks from the day of shRNA injection. (B, C) Upon termination of the experiment at post-treatment week 8, the mice were sacrificed, and the tumors were excised, photographed and weighed. (D) Tumor tissues were fixed, paraffin embedded, sectioned and stained with hematoxylin and eosin (H&E) for histological examination. (E) TUNEL assay was performed to detect apoptotic cells in the tumor tissues. (F) Expression levels of YB-1 and Cyclin-D1 in the tumor tissues were assessed by Western blot analysis. D-F show representative images from all the mice examined. Values are expressed as mean ± standard deviation. Compared with PBS control, ***P<0.001.