Immunohistochemical (IHC) staining
Brain glioma tissue and paired normal tissue microarray chips were purchased from Shanghai Outdo Biotech Co. Ltd. (Cat. # HBraG180Su01). Meanwhile pathological characteristics of these clinical samples were collected. All patients signed the informed consent form.
Briefly, after tissue sections were deparaffinized, repaired and blocked with citric acid antigen, they were incubated with anti-PSMC2 (1:100, SANTA CRUZ, Cat # sc-166972) and anti-E2F1 (1:100, Abcam, Cat # ab179445) respectively, overnight. After elution with PBS, secondary antibody IgG (1: 400, Abcam, USA, Cat # ab6721) was added and incubated. Tissue sections were then stained with DAB and hematoxylin for visualization. All tissues in the chip were pictured with microscopic and all slides were viewed with ImageScope and CaseViewer. IHC scores were determined by staining percentage scores (classified as: 1 (1%-24%), 2 (25%-49%), 3 (50%-74%), 4 (75%-100%)) and staining intensity scores (scored as 0: Signalless color, 1: brown, 2: light yellow, 3: dark brown). To distinguish between high and low expression, the median was selected as cut off-value to reduce the impact of outliers.
Human glioma cell lines U87 and U251 were purchased from BeNa Technology. U87, U251 cells were cultured in CM1-1 medium containing 90% DMEM-H medium supplemented with 10% FBS. All culture medium was changed every 3 days and cells were humid maintained in a 37°C 5% CO2 incubator.
Target gene knockdown cell models
Short hairpin RNAs (shRNA) of human PSMC2 and E2F1 and related control sequence were designed by Shanghai Bioscienceres, Co., Ltd. for the knockdown experiment. The target sequences were inserted into BR-V-108 vector using the T4 DNA ligase enzyme (NEB). Plasmids were extracted by EndoFree Maxi Plasmid Kit (Tiangen) and qualified plasmid was packaged with 293T cells. U251 and U87 cells at a density of 2×105 cells/ml were seeded in a six-well plate. 24 h later, cells were infected with 100 µL lentiviral vectors (1×107 TU/well) additive with ENI.S and polybrene (10 µg/ml, Sigma-Aldrich). After cultured at 37°C with a 5% CO2 for 72 h, the fluorescence was observed by microscope.
U87 and U251 cells with shPSMC2, shE2F1, shPSMC2+shE2F1, and the corresponding negative controls grown in the 6-well plate were harvested with TRIZOL reagent (Invitrogen, Carlsbad, USA) and then provided by the manufacturer with a standard procedure to isolate the total RNA. Next, RNA samples were washed with ethanol and then dissolved in DEPC H2O. Reverse transcription was applied to M-MLV reverse transcriptase and RNase inhibitor (Promega Corporation, Madison, Wisconsin, USA). qPCR was performed using SYBR Master Mix (Takara, Japan), where GAPDH was used as the internal control. Primer information was shown in the Table S1. Finally, the 2−ΔΔCq method was used for quantification and dissolution curve was drawn.
U87 and U251 cells with shPSMC2, shE2F1, shPSMC2+shE2F1, and the corresponding negative controls grown in the 6-well plate were harvested with Radio Immunoprecipitation Assay (RIPA) buffer (Beyotime, Shanghai, China). Proteins were degenerated in Sodium dodecyl sulfate (SDS) sample buffer, followed by separating on SDS-polyacrylamide gelelectrophoresis (PAGE) gel using electrophoresis. After that, proteins were transferred onto polyvinylidene difluoride (PVDF) membrane, and incubated with primary antibodies (Table S2) overnight at 4°C. Membranes were then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies IgG (Goat Anti-Rabbit) at room temperature for 1 h. Protein expression was then determined using ECL kit (Thermo Fisher Scientific, NY, USA).
Cell viability was measured using 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-h-tetrazole bromide (MTT) for U87 and U251 cells with shPSMC2 and the corresponding negative controls for 48 h. Briefly, cells were plated at 2×103 per milliliter. MTT solution 5 mg/ml was added to each well of the 96-well plates for 4 h. After incubation, 100 μl dimethyl sulfoxide (DMSO) were added to each well. The formazan was then quantified by measuring the 490 nm absorbance.
Colony formation assay
U87 and U251 cells with shPSMC2, shE2F1, shPSMC2+shE2F1, and the corresponding negative controls were trypsinized, resuspended, counted, and seeded in 6-well plate (2 ml/well) and incubated for 8 days to form colonies. Subsequently, cells were fixed by paraformaldehyde. After Giemsa staining, residue was washed multiple times and finally photographed with a digital camera. Numbers of colonies (> 50 cells/colonies) were counted under a microscope (MicroPublisher 3.3 RTV; Olympus, Tokyo, Japan). All determinations were repeated three times.
Cell apoptosis and cycle assay
U87 and U251 cells with shPSMC2, shE2F1, shPSMC2+shE2F1, and the corresponding negative controls cells were inoculated in a 6-well plate until cell density reached 85%. Cells were harvested, centrifuged (1200 × g), and resuspended. For cell apoptosis, 10 μl Annexin V-APC (eBioscience) was added and incubated at room temperature without light for 10 min. For cell cycle, cells were stained by PI staining solution (BD Biosciences). Apoptosis analyses and cell cycle distribution was detected Guava easyCyte HT FACS Calibur. (Millipore).
Celigo cell counting assay
After U251 cells with shPSMC2, shE2F1, shPSMC2+shE2F1, and the corresponding negative controls were cultured in 96-well plate 5 days. After plating, numbers of cells were evaluated using Celigo® image cytometer (Nexcelom, Lawrence, Mass, USA) by scanning green fluorescence every day for 5 days at room temperature.
After transfected with shPSMC2, shE2F1, shPSMC2+shE2F1, and the corresponding negative controls 72 h of U251 cells (2 × 103 cells/well) were scraped a monolayer at the scheduled time (0 h, 24 h, 48 h) for wound-healing assay and under 5% CO2 incubated at 37°C.
Suspension of U251 cells transfected with shPSMC2, shE2F1, shPSMC2+shE2F1, and the corresponding negative controls were adjusted to 5 × 105 cells/ml, and then the cells were seeded in Transwell (Corning, NY, USA). The cells were fixed with 4% paraformaldehyde and then stained with 0.1% crystal violet solution. Finally, the cells passing through the filter were photographed by an inverted fluorescence microscope.
Human apoptosis antibody array
Differentially expressed apoptosis-related proteins in U251 cells with shPSMC2, and the corresponding negative controls were detected by human apoptosis antibody array (RayBio, Norcross, GA, USA). First, the membrane was placed in the 8-well trays provided in the kit. Incubations with sample and Biotin-conjugated Anti-Cytokines should be performed overnight at 4°C. Overnight blocking and wash steps are useful for reducing background signal intensities even with completed membranes. Wash with Wash Buffer II, followed by repeating incubation with Streptavidin-HRP and chemiluminescent detection. Finally, Gelpro Analyzer software (Media Cybernetics, Rockville, MD, USA) was used to analyze protein expression levels.
Mice xenograft model
The animal experiments performed have been approved by the Animal Protection and Use Committee of Sir Run Run Shaw Hospital. Nude mice (BALB/c males, 4 weeks old) were purchased from Shanghai Lingchang Experimental Animal Co., Ltd (Shanghai, China) and were pathogen-free. Twenty mice were randomly divided into two groups (shCtrl group and shPSMC2 group) before the experiments. Meanwhile, 2 × 106 U87 cells with or without knockdown of PSMC2 suspended in PBS were injected into mice under the right axillary skin to construct a mouse xenograft model. Cultured for another 20 days after injection and collected data from 10 days after injection. Mice were injected with cells 10 days after starting to collect data twice a week, including animal weighing, measuring tumor length short diameter. The mice were finally sacrificed by injection of pentobarbital sodium, and the tumor was removed for photographing and weighing.
Repaired and blocked with citrate antigen after tumor tissue was removed from sacrificial mice. Antibody Ki67 (1:200, Abcam, USA, cat # ab16667) was added to shPSMC2 or shCtrl, respectively. After PBS elution, secondary antibody IgG (1:400, Abcam, USA, cat # ab6721) was incubated at room temperature. Tissue sections were first stained with DAB and then with hematoxylin. Finally, images were collected and analyzed by optical microscope.
RNA sequencing analysis
First, RNA was extracted and tested for quality as described above. And then, RNA sequencing analysis was performed by Genechem (Shanghai, China). Library for RNA sequencing was constructed from TruSeq Stranded mRNA LT Sample Preparation Kit (Illumina, San Diego, California, USA) according to the instructions of manufacturer, and scan it by Affymetrix Scanner 3000 (Affymetrix, Santa Clara, California, USA). DEGs were determined between the two groups based on thresholds of |Fold Change| ≥ 2.0 and FDR < 0.05. IPA (Qiagen, Hilden, Germany) based on all DEGs to analyze rich functional annotations. Z-score ≥ 2 meant that the pathway was significantly activated; otherwise the pathway was significantly inhibited.
All experiments were performed in triplicate and data were shown as mean ± SDs. Statistical analyses and graphs were performed by GraphPad Prism 6.01 (Graphpad Software) and P value < 0.05 as statistically significant. The significance differences between groups were determined using the two-tailed Student’s t test or One-way ANOVA analysis. PSMC2 expression in glioma tissues and normal tissues revealed in IHC assay were analysis with Sign test. Mann-Whitney U analysis and Spearman rank correlation analysis were used while explaining the relationships between PSMC2 expression and tumor characteristics in patients with glioma.