Cell culture
Experimental analyses were carried out in vitro using the following cell lines: Normal human astrocytes (NHA) (KG578, KeyGEN, Nanjing, China), A172 and U251 cells (HNC241, HNC1088, FDCC, Shanghai, China), and LN229 cell (the First Affiliated Hospital, Army Medical University). NHA, A172, U251, and LN229 cells were cultured in Dulbecco’s modified Eagle medium (DMEM, HyClone) supplemented with 10% (v/v) fetal bovine serum (FBS, 10270, Life Technologies), 4 mM glutamine, 100 IU/mL penicillin, 100 μg/mL streptomycin and 1% nonessential amino acids (Thermo, Carlsbad, CA, USA). All cell lines were cultured in a 37 °C, 5% CO2 incubator and passaged for less than 2 months after thawing.
CRISPR/Cas9-mediated Dazl knockdown
According to the protocol of Ran et al [27], CRISPR/Cas9 gene-editing technology was used to mediate Dazl knockdown in GBM cells. To generate Dazl-silenced cells using CRISPR-Cas9 gene-editing technology, two different short guide RNAs (sgRNAs) against DAZL were bought from Sigma (Clone ID: HS5000028071 and HS5000028072). The Dazl-sgRNAs sequences are: GCTGATGAGGACTGGGTGCTGG; GAAGCTTCTTTGCTAGATATGG. The Dazl sgRNAs were cloned into a CRISPR/Cas 9-Puro vector: hU6-gRNA-PGK-Puro-T2A-BFP. GBM cells were transfected with CRISPR plasmids and the lenti-cas9 pSpCas9(BB)-2A-GFP (PX458) plasmid (Addgene plasmid #48138) using X-tremeGENE 9 DNA Transfection Reagent (6,365,787,001, Sigma-Aldrich, USA). Lenti-Cas9 and Dazl sgRNA plasmids were transfected at a ratio of 150 ng to 50 ng per well. Puromycin (60210ES25, Yeasen Biotech, China) and blasticidin (15,205, Sigma-Aldrich, USA) selection were performed followed by the transfection. Positive clones were isolated by a medium gradient dilution method, finally confirmed by sequencing. Then Dazl deletion was further verified by Western blotting using anti-Dazl (ab34139, Abcam, USA).
Western blotting
GBM cells and tissues were harvested and lysed in RIPA lysis buffer (P0013B, Beyotime, China) supplemented with phenylmethanesulfonyl fluoride (PMSF, 1 mM, ST506, Beyotime, China) cocktails. Proteins (25 μg / well) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electro-transferred to a polyvinylidene fluoride membrane (Millipore, Bedford, UK). The membrane was blocked with 5% nonfat milk, blotted with primary and secondary antibodies. The immune reaction was detected with an enhanced chemiluminescence substrate (Thermo, USA) using a chemiluminescence imaging system (Clinx, Shanghai, China). Band density was statistically analyzed with ImageJ software. The antibodies used to detect protein expression are shown above.
RNA isolation and RT-PCR
Total RNA from GBM cells was collected using the Trizol reagent (15,596,018, Thermo, USA) and RNA quantification was done using a NanoDrop2000 spectrophotometer (Thermo, USA) by detecting absorbance at 260 and 280 nm. Subsequently, reverse- transcription of total RNA (500 ng) was performed using a PrimeScript™ RT reagent kit (RR036, Takara, Japan). Quantitative RT-PCR was performed using SYBR premix (RR820, Takara, Japan) and performed on the ABI 7500 system (Life, USA). mRNA expression was normalized to the average of human GAPDH. All reactions were performed in triplicate, and the RNA level was analyzed via the 2−ΔΔCt method. The primers used for detecting gene expression were human Dazl-F: GGTGTCGGGCGCATGTAAT; human Dazl-R: CTTTGGACACACCAGTTCGAT; human GAPDH-F: TGCACCACCAACTGCTTAGC; human GAPDH-R: GGCATGGACTGTGGTCATGAG.
Immunohistochemistry
Immunohistochemistry for Dazl was done on paraffin tissue array sections. Slices were deparaffinized by incubating in xylene and rehydrated in an ethanol gradient with decreasing amounts of ethanol until the final wash, which was water. After antigen retrieval in sodium citrate-hydrochloric acid buffer (pH 6.0, C8532, Sigma, USA), subsequent steps were to quench endogenous peroxidase activity with a 3% H2O2 solution. After blocking the sections with 10% goat serum (ab7481, Abcam, USA) for 1 h, the slides were incubated with monoclonal rabbit anti-Dazl antibodies at 4 °C overnight. Next day remove the slices from 4 °C and rewarming at RT 30 min, then all slides were incubated with HRP secondary antibodies and stained with a DAB kit (ab64238, Abcam, USA) and with hematoxylin solution (MHS1, Sigma, USA). Finally, dehydration was performed in 85, 95, and 100% ethanol and distilled water sequentially.
Cell proliferation assay
According to the manufacturer’s instructions, GBM cells were all planted with a density of 1 × 103 cells per well in 96-well plates. Following the 7 consecutive days culture, each well was replaced with 100 μl fresh DMEM containing 10 μl CCK-8 solution (CK04, DOJINDO, Japan), and incubated at 37 °C for 2 h. The optical density was measured at 450 nm on a microplate reader (Biotek, USA). Background signal was subtracted, all values were repeated 4 times.
Alkaline phosphatase staining
GBM WT cells and Dazl-knockdown cells were washed with 100 mM Tris-HCl buffer (pH 8.2). For phosphatase activity reaction, cells were treated with a Vector® Blue Alkaline Phosphatase (Blue AP) Substrate kit (SK5300, Vector Laboratories, USA) according to the manufacturer’s instruction. After staining, randomly selected 10 microscopic fields (200 × magnification) for each treatment and counted stain-positive colonies.
Cell migration and invasion assay
For cell migration assay, GBM cells (5.0 × 104 cells / well) were seeded into the upper chambers of wells in 24-well plates that had 6.5 mm polycarbonate membranes with an 8 um pore size (3422, Corning, USA). For the cell invasion experiment, Matrigel matrix (354,234, Coring, USA) in DMEM (1:3) was coated into the upper chambers. The DMEM was removed carefully when the Matrigel matrix was solidified 12 h later. A total of 5.0 × 104 cells suspended in serum-free DMEM were seeded into the upper chambers. DMEM with 10% FBS was added to the lower chambers. Twenty-four hours later, cells remaining on the upper surfaces of the membranes were removed, with the others that invaded through the membrane filters being fixed with methanol for 30 min, stained with crystal violet (C1021, Beyotime, China) for 30 min, and photographed.
In vivo experiments: xenograft model
All animal experiments complied with the “Guide for the Care and Use of Laboratory Animals” of the National Institutes of Health and all animal experiments adhered to the ARRIVE guidelines. To explore whether Dazl is involved in the tumorigenicity of glioblastoma in vivo, Dazl knocked-down cells (1.5 × 105) and GBM WT cells (1.5 × 105) were subcutaneously injected into 4-week-old female BALB/c nude mice (n = 6 per group, Shanghai Lab. Animal Research Center, China) in their back. Vernier calipers were used to measure the tumor diameter of nude mice every 6 days to assess tumor growth. Tumor volumes were calculated according to the formula: V (mm3) = L × W2 / 2 (where V is the tumor volume, L is the length, and W is the width). The survival of the remaining mice was assessed via Kaplan-Meier analysis. The mice were euthanized via CO2 at the end of the experiments. Tumors from each mouse were removed, photographed, measured, and weighed, then were used for biochemical (frozen tissue) and histological (paraffin fixed tissue) analyses.
Statistical analysis
Statistical analysis was carried out by using GraphPad Prism version 6.0 (San Diego, CA, USA). Each figure shows an accurate representation of the error bars. Unless otherwise specified, all experiments were performed at least in triplicate. P < 0.05 were considered as statistically significant.