2.1 Cell lines and cell culture
The human Microglia Clone 3 (HMC3) cell line (CRL-3304), the human glioma U251 cell line and the U87 cell line (HTB-14) were obtained from the American Type Culture Collection (ATCC). The U251 and U87 cell lines were authenticated using Short Tandem Repeat (STR) analysis. U251 and HMC3 cells were cultured in high glucose DMEM medium (Gibco, USA) with 10% fetal bovine serum (FBS, Biological Industries, Israel), whereas U87 cells were cultured in low glucose DMEM medium (Gibco, USA) with 10% FBS. All cells were cultured at 37°C under humidified atmosphere containing 5% CO2.
2.2 Lentivirus Package and infection
The GeCKO library was acquired from Addgene (www.addgene.org/crispr/libraries/). GeCKO library plasmids, pVSVg (AddGene, USA) and psPAX2 (AddGene, USA) were added to 100µL Opti-MEM at a ratio of 1:0.5:1.5, and then the mixture with lipofectamine 2000 (Invitrogen, USA) was incubated for 20min and added into HEK293T cells. After 48h, the cell supernatant containing lentiviruses was collected and the virus titer was 3E + 8Tu/mL. Cells were seeded in a 10cm dish at 70% confluence, to which 10µL lentivirus and 10µL polybrene (1000×) were then added to constitute 10mL complete medium. The complete medium was then slowly added to U251 and U87 cells. The lentivirus-containing medium was replaced with complete medium after 48h. Infected cells were screened with 2µg/mL puromycin for 14d.
2.3 Sample preparation
After being screened by puromycin, the survival cells were treated with 1000µM TMZ for 72h. Alive cells were then collected to extract DNA using Blood & Cell Culture Midi Kit (Promega, USA). PCR was used for amplification subsequently. The forward primer sequence for amplifying lentiCRISPR sgRNAs was 5’-CTTGTGGAAAGGACGAAACA-3’, the reversed primer was 5’-GCCAATTCCCACTCCTTTCA-3’. The PCR conditions were as follows: 95℃-5min; 95℃-30s, 54℃-30s, 72℃-30s for 35 cycles; 72℃-5min; 4℃-hold. EasyPure Quick Gel Extraction Kit (TransGen Biotech, China) was used to extract amplicons from 1% agarose gels, and then were sent to company for NGS (Novogene, China). Based on information obtained from sequencing, sgRNA of Top Hits was sorted.
2.4 Plasmids and small interfering RNA (siRNA) transfection
siRNA of candidate genes CD99L2, ALKBH3, and PPAPDC3 were synthesized using RIBOBIO (China). Sequences of siRNAs are listed in Table 1. Transfections were undertaken on lipofectamine RNAiMAX Reagent. Plasmid CD99L2 and its negative control were constructed using Genechem (China). Transfections were performed on lipofectamine 2000 Reagent as directed in the manual.
Table 1
siRNA sequences of candidate genes for knockdown
Gene
|
Sequences
|
CD99L2
|
si-1: 5’-GCAGTGAAAGAAACTTCCT-3’
|
|
si-2: 5’-GTCTCAACGCAGACTACGT-3’
|
ALKBH3
|
si-1: 5’-GCCACGAGTGATTGACAGA-3’
|
|
si-2: 5’-CGAGAGTGAACCTGACCTT-3’
|
PPAPDC3
|
si-1: 5’-CTGGCCATCGATATCTGTA-3’
si-2: 5’-TCAAGGGCATCGCCTTCAA-3’
|
2.5 RNA extraction and Real-time PCR
Total RNA was extracted from differently treated cells using Trizol reagent (TaKaRa, Japan). cDNA was synthesized from 1µg RNA using Prime Script RT reagent Kit with gDNA Eraser (TaKaRa, Japan). Quantitative RT-PCR was then undertaken using 2×SYBR Green qPCR Master Mix (TaKaRa, Japan) in the Roche LightCycler480. GAPDH was used to normalize mRNA expression. Primers used for qPCR are listed in Table 2 (BioSune, China). Related expression of candidate genes was analyzed using 2−ΔΔCt.
Table 2
Primers of candidate genes for qPCR
Gene
|
Sequences
|
CD99L2
|
F: 5’-TGACAAGGGTAAAGGTGATGG-3’
|
|
R: 5’-AACTTCTTCTGCTGGTAGGAG-3’
|
ALKBH3
|
F: 5’-GCCACGAGTGATTGACAGAG-3’
|
|
R: 5’-AAAGCCAGGATACAAACAGACC-3’
|
PPAPDC3
IL2RG
STEAP4
EDARADD
GAPDH
|
F: 5’-GACATCTACGCCTTCCC-3’
R: 5’-GCCCAGAGCACCAGCAG-3’
F: 5’-GTGCAGCCACTATCTATTCTCTG-3’
R: 5’-GTGAAGTGTTAGGTTCTCTGGAG-3’
F: 5’-GGCTTTGGGAATACTTGGGTT-3’
R: 5’-TGGACAAATCGGAACTCTCTCC-3’
F: 5’-CCATTCAAGATACGGAACTCCC-3’
R: 5’-AGCAAGTCACTTATGGTGGGG-3’
F: 5’-CCCATCACCATCTTCCAGGAG-3’
R: 5’-GTTGTCATGGATGACCTTGGC-3’
|
2.6 Cell viability assay
After transfection, U251 or U87 cells were seeded in 96-well plates at a density of 5× 104 cells/mL. U251 cells were treated with TMZ (Selleck, USA) at concentrations of 0, 300, 600, 900, 1200, 1500, 1800, and 2100µM for 48h, whereas U87 cells were treated with TMZ at concentrations of 0, 200, 400, 600, 900, 1200, 1500, and 1800µM for 48h. Cell viability was detected using CCK-8 reagent (Bimake, USA) according to the protocol. CCK-8 is an indicator of redox reaction. In the presence of electronic coupling reagents, dehydrogenase in living cells can be used to catalyze the production of yellow formazan dye from tetrazolium salt WST-8. The amount produced has a linear relationship with the number of living cells. Optical density (OD) was read at 450 nm on a multi-mode reader (Synergy LX, BioTek, USA). Half maximal inhibitory concentration (IC50) was computed using GraphPad Prism 7.0 (GraphPad Software, CA).
2.7 Flow cytometry analysis of apoptosis
TMZ-induced apoptosis in GBM cell lines was detected using Annexin V-FITC apoptosis detection kit (Beyotime, China). U251 or U87 cells were seeded in 6-well plates at a density of 1.5×105 cells/mL, transfected with siCD99L2 and siNC for 48h, and treated with 1000µM TMZ for another 48h, with DMSO serving as negative control. Cells were collected and washed thrice using PBS, 195µL Annexin-V-FITC binding buffer was added for 1×104 cells, and 5µL Annexin-V-FITC as well as 10µL PI were added into cells and incubated for 10-20min at room temperature in the dark. Processed samples were analyzed using flow cytometer (FACSCalibur, BD Biosciences) within 1h. During this time, the cells were placed in ice bath.
2.8 Western Blot
Cells were lysed with RIPA lysis buffer and protease inhibitors (Beyotime, China) to extract the total protein extraction. The protein concentration was determined by the bicinchonininc acid (BCA) protein assay kit (Beyotime, China). 20µg/lane of protein were subjected to 12% SDS-polyacrylamide gel electrophoresis and then transferred to PVDF memebrane. Membranes were blocked with 5% not-fat milk for 1.5h, then incubated with primary antibodies overnight at 4℃, then incubated with the secondary antibodies at room temperature for 1h. The primary antibodies used were anti-CD99L2 (1:1000, ABclonal, A15907) and anti-β-Tubulin (1:10000, Sigma, T8328), the secondary antibodies were Rabbit Anti-Mouse IgG H&L (1:10000, abcam, ab6728) and Goat Anti-Rabbit IgG H&L (1:10000, abcam, ab205718). All experiments were performed at least three times.
2.9 Cell Proliferation and Clone formation assays
The EdU assay kit (RIBOBIO, China) was applied to test cell proliferation detection, in which the thymidine analog EdU was integrated into DNA during proliferation stage. Cells from different groups were seeded in 96-well plate at densities of 3000 cells/well. Cells were labeled by 100µL EdU (5µM) for 2h. Subsequently, the cells were fixed in 4% paraformaldehyde for 30min, stained newly synthesized DNA into red fluorescence by 100µL 1×Apollo® for 30min, and then dyed nucleus into blue fluorescence by 100µL 1×hoechest for another 30min. Finally, fluorescence microscope (EVOS M7000) was used to detect EdU via Apollo® based fluorescent dyes, and Image J software was used to calculate the number of cells in the field.
Transfected U251 or U87 cells were seeded in 96-well plates at densities of 2000 cells/pole. A total of five plates were seeded. CCK-8 reagent was used to detect OD value at 450 nm for five consecutive days and a cell proliferation curve was constructed. Each experimental and control group had six multiple holes.
For plate clone formation assay, different groups of cells (1000 cells/well) were seeded in 6-well plates. The culture medium was changed every third day and cultured for two weeks. On the fourteenth day, cells were fixed using paraformaldehyde for 30min and dyed using crystal violet solution for another 30min. After scaning and photograph the whole-field with fluorescence microscope, and the number of formed cloned was recorded with Image J software.
2.10 Scratch wound assay
Wounds were created when confluence of differently treated cells reached 90% or more. A 200µL pipette tip was used to make a scratch wound. The culture medium was superseded using Serum-free opti-MEM. Photographs were taken at 0, 24, and 48h to record cell migration.
2.11 Transwell migration and invasion assay
Transwell chambers (8µm, Corning, NY) were used to detect cell migration or determine cell invasion with Matrigel. For transwell migration assay, 600µL culture medium with 20% FBS was added into lower chamber and 200µL cell suspension (contained roughly 3×104 cells) with serum-free medium was added into upper chamber. For transwell invasion assay, 80µL of Matrigel and serum-free medium (1:8) was added into upper chamber to incubate for 6h in a 37℃ cell incubator. The subsequent procedure was the same as the migration assay. After incubating for 48h, cells were fixed using paraformaldehyde for 30min and dyed using crystal violet solution for another 30min. The number of stained cells was computed in 5 random fields and photographs were taken using a microscope camera.
2.12 Immunohistochemical (IHC) staining and evaluation
Formalin fixed paraffin-embedded (FFPE) tissues were prepared into sections of 3µm. The slides were dewaxed and dehydrated with gradient alcohol. Then, slides were immersed in the 95℃ antigen repair solution for 20min and cooled to room temperature. After blocking by the goat serum for 1h, slides were incubated with CD99L2 antibodies (1:20, ab224164, Abcam, USA) overnight in an incubator at 4℃. And then incubated with Goat anti-Rabbit IgG secondary antibodies (1:500, ab205718, Abcam, USA). 3,3-diaminobenzobutyl (DAB) was used to stain the nuclei in the dark. Finally, slides were covered with neutral balsam and photographed with a microscope.
IHC score was evaluated in terms of both intensity and percentage of immune-reactive cells in five random fields. Staining intensity was scored as follows: no staining: 0, primrose yellow staining: 1, intermediate positive staining: 2, brown staining: 3. The percentage of CD99L2 positive cells were scored as follows: <5%: 0, 6%-25%: 1, 26%-50%: 2, 51%-75%: 3, > 75%: 4. The final IHC score was calculated as staining intensity + percentage of positive cells. We defined score 0–3 as low expression and score 4–7 as high expression. IHC scores were blind-checked by two pathologists separately.
2.13 Patients and tissue samples
A total of 55 FFPE GBM tissues and clinical data were collected for retrospective analysis from Xiangya Hospital during February 2015 to December 2018. This study protocol was approved by the Joint Ethics Committee of the Central South University Health Authority. Informed consents were waived due to the retrospective nature of this study.
All patients received radiotherapy and TMZ treatment after surgery. FFPE tissues included in this study were the samples of patients with GBM IV grade. Samples of patients lacking complete PFS information were excluded and those with other cancers or underlying diseases that may affect the treatment were also excluded. Information about methylation status of MGMT promoter was available for 54.5% of patients.
Progression-free survival (PFS) was calculated as the duration, in months, from the date of initial surgical resection to the date of recurrence, which was defined by magnetic resource imaging (MRI).
2.14 Statistical analyses
The general statistics analyses were conducted using SPSS V.25 software (SPSS Inc, Chicago, IL). Unpaired 2-tailed Student’s t-test or analysis of variance (ANOVA) were used for continuous variables. Pearson’s χ2 was performed for categorical variables. To draw the survival curve, Kaplan-Meier analysis and the log-rank test were utilized. All data were expressed as mean ± standard deviation (SD). P < 0.05 was considered statistically significant.