NSCs culture, neural differentiation and transfection
This study was approved by the Animal Care and Use Committee of the First Affiliated Hospital of Zhengzhou University. Primary NSCs, obtained from 13.5 days embryos of Wistar rats, were maintained in the growth medium supplemented with 20 ng/mL human epidermal growth factor (hEGF; Gibco, Grand Island, NY, USA), 10 ng/mL basic fibroblast growth factor (bFGF; Gibco), 1% N2 (Gibco), 100 U/mL penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA) at 37 °C in a humidified atmosphere of 5% CO2. Neurospheres were digested using 0.25% trypsin into single cell suspension to get clone neurosphere. To induce neural differentiation, neurospheres were seeded onto poly-l-lysine/laminin (invitrogen)-coated coverslips at a density of 2 × 104 cells/coverslip. Subsequently, NSCs were cultured in DMEM-F12 (1:1) differentiation medium supplemented with 1% N2 (Gibco), 2% B27 (invitrogen), 0.5% fetal bovine serum (FBS; Gibco), 20 ng/mL of neurotrophin-3 (NT-3) (PeproTech, Rocky Hill, NJ, USA), 20 ng/mL of brain-derived neurotrophic factor (BDNF) (PeproTech), 10 ng/mL of leukemia inhibitory factor (LIF) (R&D Systems, Inc., Minneapolis, MN, USA), and 2 mM of l-glutamine (Gibco) for 7 days. The differentiation medium was renewed every other day.
To overexpress DLL4, the full-length of DLL4 sequence was synthesized with the sequence released in Genebank (Accession number: NM_019074.2) as a template and inserted to the BamHI and XhoI sites of empty pcDNA3.1 vector (invitrogen), named as pcDNA-DLL4. miR-124 mimic (miR-124) (5′-UAA GGC ACG CGG UGA AUG CC-3′), miRNA scrambled control (miR-control) (5′-UUC UCC GAA CGU GUC ACG UTT-3′), miR-124 inhibitor (anti-miR-124) (5′-UAA GGC ACG CGG UGA AUG CC-3′), inhibitor scrambled control (anti-miR-control) (5′-CAG UAC UUU UGU GUA GUA CAA-3′), siRNA targeting DLL4 (si-DLL4), and siRNA scrambed control (si-control) were purchased from the GenePharma (Shanghai, China). The oligonucleotide sequence of si-DLL4 was as follows: 5′-CCT CTC CAA CTG CCC TTC AAT TTCA-3′. NSCs were seeded onto a six-well plate at a density of 2 × 105 cells/well 24 h prior to transfection. Subsequently, NSCs were transfected with 20 nM miRNAs, 40 nM siRNAs or 200 ng pcDNA plasmids using the Lipofectamine 2000 reagent (invitrogen).
Neurosphere assay was performed to determine the self-renewal ability of NSCs. After introduction with miR-124, miR-control, si-DLL4, si-control, miR-124 + vector, or miR-124 + pcDNA-DLL4, dissociated neurospheres were seeded at a density of 5000 cells in 96-well plates in NSC medium. After 2–3 weeks of incubation, the image fields were captured covered roughly a 3.75 mm2 area using an Olympus inverted light microscope (Olympus, Tokyo, Japan) at 200× magnification. The total number of neurospheres with a diameter of > 50 μm was counted and the averaged diameter of neurospheres was measured with ImageJ software (NIH, Bethesda, MD, USA).
Quantitative real-time PCR
Total RNA was extracted from NSCs using TRIzol reagent (invitrogen) based on the instruction specified by the manufacture. For miR-124 expression detection, aliquots of total RNA (0.5 μg) was reversely transcribed into cDNA using the one-step Primescript miRNA cDNA synthesis kit (Takara, Dalian, China) and RT-PCR was performed using the TaqMan MicroRNA Assay kit (Applied Biosystems Inc., Foster City, CA, USA). For DLL4 mRNA expression evaluation, the first strand of cDNA was synthesized from total RNA using M-MLV reverse transcriptase (Clontech, Palo Alto, CA, USA) and RT-PCR was carried out by the SYBR Green I Real-Time PCR kit (Takara). Real-time PCR was then executed with an ABI7300 Real-Time PCR System (Applied Biosystems Inc.). Relative gene expression levels of miR-124 and DLL4 were calculated using the 2−ΔΔCt method, with U6 and GAPDH as respective internal control.
Protein (50 µg per lane) exacted from NSCs was electrophoresed in 12% SDS-PAGE (Beyotime Institute of Biotechnology, Shanghai, China) and then transferred onto polyvinylidene difluoride membrane (PVDF; Amershame, GE, Buckinghamshire, UK). Following blocked with 5% non-fat milk for 1 h, the membranes were incubated overnight at 4 °C with primary antibodies (Abcam, Cambridge, MA, USA) against DLL4, ki-67, β-tubulin, GAFP, HES1, HEY2, cyclin D1 (CCND1), and β-actin. The membranes were subsequently blotted with HRP-labeled secondary antibody (Santa Cruz Biotech, Santa Cruz, CA, USA) for 1 h at room temperature. The immunoreactive bands were detected using the enhanced chemiluminescence system (Millipore, Billerica, MA, USA).
Cell proliferation assay
NSCs (5 × 104 cells/well) were seeded into 96-well plates and transfected with miR-control, miR-124, si-DLL4, si-control, miR-124 + vector, or miR-124 + pcDNA-DLL4. MTT analysis was performed to detect cell viability by measuring the optical density at 490 nm at 24, 48, and 72 h posttransfection.
Luciferase reporter assay
The wild-type or mutant 3′UTR of DLL4 containing the putative miR-124 binding sites was synthesized and inserted into pGL3-luciferase reporter plasmids (Promega, Madison, WI, USA), named as DLL-3′UTR-WT or DLL-3′UTR-MUT. The mutated 3′UTR of DLL4 was generated using QuickChange Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA, USA). Then, NSCs were transfected with DLL-3′UTR-WT or DLL-3′UTR-MUT reporter, pRL-TK (Promega), and miR-124 or miR-control using Lipofectamine 2000 (invitrogen). Cells were harvested 48 h after transfection for luciferase activities measurement with a Dual-Luciferase Reporter Assay system (Promega). Renilla luciferase activity was used as the normalization for firefly luciferase activity.
Primary NSCs and their differentiation cells were plated onto coverslips, fixed with 4% paraformaldehyd and permeabilized using 0.3% Triton™X-100 in PBS buffer for 1 h. After blocked with goat serum (10%), cells were incubated with the primary antibodies against GFAP, β-tubulin-III and Nestin (Sigma-Aldrich) at 4 °C overnight, followed by incubation with an Alexa Fluro 568-conjugated secondary antibodies (Santa Cruz Biotech, Santa Cruz, CA, USA) for 1 h. Cell nuclei were counterstained with 1 μg/mL DAPI (Sigma-Aldrich). Slides were analyzed at room temperature on a Zeizz AX10 microscope (Carl Zeiss, Thornwood, NY, USA) using Image J (NIH, Bethesda, Maryland, USA).
All data are presented as mean ± standard deviation (SD) from three independent experiments. All statistical analyses were performed using SPSS 11.7 software (SPSS Inc., Chicago, IL, USA) with two-tailed Student’s t test or one-way analysis of variance (ANOVA). For all results, differences were considered as statistically significant when P value was less than 0.05.