2.1 Cell culture and treatment
Human glioma cell line U251 was obtained from American Type Culture Collection (ATCC) (Manassas, VA, USA). U251 cells were cultured in a standard humidified incubator (5% CO2; 37 °C) with RPMI-1640 (Invitrogen, Carlsbad, CA, USA) culture medium which supplemented with 10% fetal bovine serum (FBS, Carlsbad, CA, USA) and 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA).
For treatment, as previously described [28], briefly, U251 cells were irradiated by a linear accelerator 6-MV X-rays with the dose rate of 4 Gy/min. The procedure was performed daily for 5 months, starting with 1 Gy/fraction and ending with 10 Gy/fraction. The X-ray treated U251 cells were labeled as U251R cells.
2.2 Quantitative real time-PCR (qRT-PCR)
Total RNA was isolated from glioma cells with an RNA extraction kit (Takara Biotechnology, Japan). Then, Reverse Transcription Kit (Takara Biotechnology, Japan) was used to obtain cDNA and reverse transcribe RNA. The primers used in this study were synthesized by Yingjun Technology (Shanghai, China) which showed in Table 1. qRT-PCR was conducted using the SYBR Green Realtime PCR Master Mix (Toyobo, Japan) and performed on ABI 7900 fast Real‐time PCR Systems (Applied Biosystems, USA). GAPDH were used as internal controls.
Table 1
Primers used in the present study
Gene
|
Primer sequence
|
LRIG1
|
forward: 5′-GAAAAGGGACTCTGGTTGGGAT-3′
|
reverse: 5′-AGGAAGTCATCGCACACGAA-3′
|
CTLA-4
|
forward: 5′-AGGTGACTGAAGTCTGTGCG-3′
|
reverse: 5′-CATGAGCTCCACCTTGCAGA-3′
|
GAPDH
|
forward: 5'-CCGGGAAACTGTGGCGTGATGG-3'
|
reverse: 5'-AGGTGGAGGAGTGGGTGTCGCTGTT-3'
|
2.3 Western blot
Total proteins from human tissue or glioma cells were extracted by using a radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime Biotechnology Co., Ltd., Shanghai, China). The protein samples were separated using 8% SDS-PAGE. After transferred onto PVDF membrane, the primary antibodies were added: anti-LRIG1 (PA5-52860, 1:500, Invitrogen, Carlsbad, CA, USA), anti-Akt (ab235958, 1:1000, Abcam, UK), anti-Akt (phospho S473) (ab81283, 1:1000, Abcam, UK), anti-CTLA-4(PA5-47547, 1:250, Invitrogen, Carlsbad, CA, USA), anti-Tubulin (ab210797, 1:1000, Abcam, UK). Tubulin served as internal control. After 24 hours of incubation, membrane was incubated along with secondary antibody immunoglobulin G (IgG) at room temperature for 2 hours.
2.4 Cell transfection
The LRIG1 was overexpressed or knockdown by using LRIG1 overexpression plasmid (Flag- LRIG1) or Short hairpin RNAs (shRNA) targeting LRIG1 (sh-LRIG1), respectively. The plasmid or empty vectors were purchased from GeneChem Corporation (Shanghai, China). Lipofectamine 3000 (Invitrogen, USA) was used for cell transfection following manufacturer’s instructions.
2.5 CCK-8 assay
Transfected U251 cells or U251R cells (2 × 105 per well) were seeded into a 96-well plate. CCK-8 Kit (Abcam, UK) and RPMI-1640 were added into each well. After incubation with CCK-8 solution for 2 hours, optical density of cells was measured at 450 nm at different time points by using micro-plate reader (Thermo Fisher, USA).
2.6 Flow cytometry assay
Cell apoptosis was detected using Annexin-V-propidium iodide (PI) apoptosis assay. Annexin V-FITC Apoptosis Detection Kit (Invitrogen, Carlsbad, CA, USA) was used according to the manufacture’s instruction. Briefly, different groups of transfected U251R cells were with propidium iodide (PI) at 37°C for 30min, followed by flow cytometry (Beckman Coulter FC500, CA, USA).
2.7 Co-Immunoprecipitation
Co-Immunoprecipitation was performed to detect the combination of LRIG1 and CTLA-4. Briefly, cells were lysed in 500 μl co-IP buffer containing a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, Missouri, USA). Then, 20 μl immobilized protein A/G beads was incubated with cell lysates for 1h at 4 °C. Primary antibody or control Ig G were incubated with lysates for 24 hours. Finally, immobilized protein A/G beads were added, then, the proteins were prepared for western blot.
2.8 Human glioma tissue collection
Tissue samples of paired glioma tissues and paracancerous tissues were collected from patients who underwent glioma resection at the second affiliated hospital of Nanchang university. All patients enrolled in this study have given their informed written consents prior to conduct the clinical research related procedure and this study was approved by the second affiliated hospital of Nanchang university Ethical Committee (No. 2019003). All patients have received 5 weeks’ radiation therapy. Tissue samples were collected and immediately snap-frozen in liquid nitrogen after surgery (-80°C) for further use.
2.9 Immunohistochemistry (IHC)
The expression of LRIG1 and CTLA-4 proteins was determined in human glioma tissues by IHC. Briefly, paraffin‐embedded sections were dewaxed and dehydrated by xylene and ethanol. Then, after incubation with 50 μl of 10% goat serum for 60min, the sections were probed with primary antibodies: anti-LRIG1 (ab197985, 1:1000, Abcam, UK), anti-CTLA-4 (ab227709, 1:1000, Abcam, UK) for 24 hours. Then, peroxidase‐labeled secondary antibody (Invitrogen, Carlsbad, CA, USA) were added and incubated for 30min. The sections were developed using diaminobenzidine (DAB).
2.10 Statistical analysis
All cell experiments were repeated triplicate. The data were exhibited as Mean ± SD. SPSS21.0 (IBM Corp. Armonk, NY) software was used for data analysis. Two-tailed Student's t-test, one-way ANOVA, Pearson correlation coefficient were used in this study. P < 0.05 was considered as statistically significant difference.