Cell lines and cell culture
Human glioma cell lines (A172, T98G, U251 and U87), human glial cell line HEB and human HEK293T cell line were obtained from American Type Culture Collection (ATCC, USA). A172, T98G, U251, U87, HEB, HEK293T cells were cultured in DMEM (Hyclone, USA) supplemented with 10% heat-inactive FBS, 100 U/mL penicillin and 100 μg/mL streptomycin. Cells were incubated in a 5% CO2 humidified incubator at 37°C. According to cell demands, the medium was refreshed in regular intervals. In brief, Tumor tissues were washed immediately after resection in HEPES-buffered saline, and homogenized and treated for 30 min with 0.025% Trypsin/EDTA solution at 37° C. The cell homogenate was filtered by 80 mm cell strainer and centrifuged at 180g for 5 min. After 2 washes with medium (DMEM, 10% FCS), the cells were seeded out for propagation and kept under differentiating conditions.
GBM sample collection
The GBM samples that used for expression level assay were collected from patients who underwent surgery at Tiantan Hospital, which were immediately frozen in liquid nitrogen after tumor resection. Informed consent of patients was obtained as approved by Ethics Committee of Beijing Tiantan Hospital, Capital Medical University (KY 2018-052-01).
Lentiviral infection
Three siRNA(siRNA-1 (788-806): GGTGGAGTGTTATGATTAT; siRNA-2 (1397-1415): GCAGACAGCTTCTCAATAT; siRNA-3 (1465-1483): CCAGACAAGCTATAGTTAA) were synthesized and cloned in pLVsiRNA. Then, to construct the overexpression plasmids, the coding sequence of human gene were synthesized and subcloned into the pcDNA3.1 lentivirus vector. Together with package plasmids, the siRNA-expressing and overexpressing constructed plasmids were transfected into 293T cells. The cells (with 80% of confluence) were infected with lentivirus supernatants, after 72h infection, the expression was determined using q-PCR and Western Blot.
CPNE3 knockdown in xenograft mice model
CPNE3 knockdown was generated in U251 cells with pLKO.1 -puro lentiviral vectors expressing shRNA against CPNE3 gene products.
shCPNE3-1 (788-806): GGTGGAGTGTTATGATTAT,
shCPNE3-2 (1397-1415): GCAGACAGCTTCTCAATAT,
shCPNE3-2 (1465-1483): CCAGACAAGCTATAGTTAA.
Oligonucleotides were amplified by PCR using the T4 DNA Polymerase (Thermo), PCR products containing shCPNE3-1, shCPNE3-2, shCPNE-3 and non-silencing shNC (controls) were subcloned into the pLKO.1-puro vectors via EcoR I and AgelI restriction sites.
Quantitative Real-time PCR (qPCR)
Total RNA of suspension cultured all cells were isolated by using the RNAiso Plus kit (invitrogen). mRNA was transcribed into cDNA with OneScript Plus Reverse Transcriptase kit (Fermentas). q-PCR was performed using SYBR Green PCR MasterMix kit (Thermo) on Real-time System (ABI). Primer sequences are as follows:
CPNE3: F 5'-CATTGTAGAGGCGTATCG-3', R 5'-CCATCACCATCCAGAAAC-3'
GAPDH: F 5'-AATCCCATCACCATCTTC-3', R 5'-AGGCTGTTGTCATACTTC-3'
The amount of PCR product for CPNE3 was normalized with the amount of GAPDH product to estimate the amount of variation between samples.
Western Blot
Cells were lysed in RIPA lysis buffer containing 1 mM phenylmethanesulfonyl fluoride (PMSF). Total protein lysates (25 μg) were fractionated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to PVDF membranes, and blocked at room temperature for 1 h with 5% nonfat dry milk and followed by overnight incubation with the anti-CPNE3 antibody (1:500, Abcam, Ab236606), anti-PCNA antibody (1:1000; Cell Signaling Technology, #13110), anti-ki67 antibody (1:500, Abcam, Ab16667), anti-XIAP antibody (1:1000, Abcam, Ab28151), anti-Bim antibody (1:1000, Abcam, Ab7888), anti-p-P13K antibody (1:1000, Abcam, Ab182651), anti-P13K antibody (1:1000, Abcam, Ab133595), anti-AKT antibody (1:1000, Cell Signaling Technology, #9272), anti-p-AKT antibody (1:2000, Cell Signaling Technology, #4060), anti-GAPDH antibody (1:2000, Cell Signaling Technology, #5174). An HRP-conjugated secondary antibody (1:1000, Beyotime) was applied for 1h at room temperature. The intensity for each protein band was corrected by the intensity of the GAPDH band and was normalized to facilitate comparisons.
Cell proliferation assay
The cells viability was assessed by Cell Counting Kit-8 (CCK8) (SAB) according to the manufacturer’s protocol. In brief, for CCK8 assay, cells were seeded in 96- well plates (3×103 cells/well) and then cultured at 37 ℃ in a humidified atmosphere with 5% CO2. After 12h, 24h, 48h and 72h incubation, respectively, 10μl CCK8 solution was added into each well. The plates were incubated for an additional 1h before detecting the absorbance value at 450 nm wavelength with a microplate reader (Perlong, Beijing).
Cell apoptosis assay
For cell apoptosis assay, cells were seeded in 6-well plates and transfected, then the dual-staining of FITC-conjugated annexin V and propidium iodide (PI) was performed as follow. At 48h post-transfection, the cells, including floating cells, were harvested and washed twice with 4 °C PBS, then resuspended in binding buffer (10 mM HEPES/NaOH, 140 mM NaCl, 2 mM KCl). Annexin V was cultured for 15 mins in the dark. Cells were then washed again, centrif uged and resuspended in binding buffer. Before flow cytometric analysis, PI was added to each sample. The apoptotic cells were quantified by cytoFLEX system, cells labeled Annexin V +/PI− were early apoptotic cells.
Nude mice xenograft study
The U251 cells were resuspended in serum-free DMEM medium at a concentration of 5 × 107 cells/mL. Eighteen male nude mice were randomly assigned to three groups, and each mouse was inoculated with 0.1 mL of cell suspension in the right axillary sub cutis. The length and width of the tumor was measured weekly using a vernier caliper, and the tumor size was calculated as volume (mm 3) = 0.5 × length (mm) × width 2 (mm 2). The mice were euthanized 5 weeks later, the tumors were collected and weighed, and the growth curve was calculated.
Immunofluorescence
The xenograft tumor tissues were incised into small pieces, normally with a size of 1.5cm×1.5cm x 0.3cm, and prepared by paraffin embedding. Generally, 5 µm sections were air-dried for 2h at room temperature. sections were washed three times in PBS and blocked in 2% BSA, 0.1% Tween20 in PBS at RT. Then, sections were washed three times in PBS and incubated with primary antibodies, Ki-67 (Abcam, Ab15580) and PCNA (Abcam, Ab18197), at 4 ℃ overnight. The following day, sections were washed three times in PBST (0.05% Tween20 in PBS) and the Alexa Fluor 488 secondary antibodies (1:500, Byotime, A0423) were used. DAPI (1:500, Byotime, C1002) were added per section and incubated for 1h in the dark. All images were taken using a Fluorescence microscope (NIKON, ECLIPSE Ni).
Histology
The xenograft tumor tissues were fixed and followed by paraffin embedding. Overall, 5 µm sections were air-dried for 2h at room temperature. Tissue sections were deparaffinized, rehydrated, and histologically stained with hematoxylin and eosin (H&E), All images were taken using a microscope (NIKON, ECLIPSE Ni).
Gene set enrichment analysis
We used Gene Set Enrichment Analysis (GSEA v2.0, available online: http://www.broad.mit.edu/gsea/) to analyze the association between expression of CPNE3 and biological processes/pathway, phenotypes. Pre-defined gene set was obtained from the Molecular Signatures Database, MSigDB (http://software.broadinstitute.org/gsea/msigdb). Samples from the TCGA datasets were divided into high- or low- CPNE3 expression groups using the median as the cutoff. Default settings were used and thresholds for significance were determined by permutation analysis (1000 permutations). False Discovery Rate (FDR) was calculated. A gene set is considered significantly enriched when the FDR score is <0.25.
Statistical analysis
Data was presented as mean of triplicates ± standard deviation (SD). Differences between groups were established by repeated-measures ANOVA followed by Bonferroni test and the Student t-test. p values < 0.05 were considered significant.