Tissue specimens
Tissue samples were retrieved from HuanHu Hospital in Tianjin, China, with a total of 5 cases of non-tumor brain tissue (traumatic brain injury) and 42 cases of glioma tissue. Include 9 cases of WHO Ⅰ grade (oligodendroglioma), 6 cases of WHO Ⅱ grade (astrocytoma), 8 cases of WHO Ⅲ grade (five of anaplastic oligodendroglioma and three of anaplastic astrocytoma), and 19 cases of WHO Ⅳ grade (GBM). The patients from whom the samples were derived did not receive any anti-cancer therapies in preoperative treatments, such as chemoradiotherapy. Samples be placed at -80°C for later use.
Cell culture
The human glioma cells (U251, LN229, U87, A172) and normal glial cells (NHA) came from the Laboratory of Medical Research Center, Affiliated Hospital of Jining Medical College. All cell lines were grown using DMEM medium (DMEM, Gibco) supplementing with 10% fetal bovine serum (FBS, Excell) and 1% penicillin-streptomycin (Gibco) in a 37°C, 5% CO2 cell culture incubator.
Cell transfection
Four different FANCI-specific small interfering RNA (siRNA) sequences (Shanghai, Genepharma) was used in this study to inhibit the expression of FANCI in glioma cells. Next, pcDNA3.1-FANCI overexpression plasmid (Shanghai, Genechem) targeting human FANCI were synthesized to promote the expression of FANCI. And the effect of transfection verified by real-time PCR and western blot.
Sequence 2267: sense (5'-3'): GCACCAGUAUUGGCAUAAATT;
antisense (5'-3'): UUUUUGCCAAUACUGGUGCTT
Sequence 1292: sense (5'-3'): GCCCAAGUCUUUAGAAUTT;
antisense (5'-3'): AUUCUAGAAAGACUUGGGCTT
Sequence 2097: sense (5'-3'): GGCCUGGUAUAAGAAUACATT;
antisense (5'-3'): UGUAUUCUAUACCAGGCCTT
Sequence 573: sense (5'-3'): CAGGUGGGAUCAGCAAUAUTT;
antisense (5'-3'): AUAUUGCUGAUCCCACCUGTT
Negative control sequence: sense (5'-3'): UUCUCCGAACGUGUCACGUTT;
antisense (5'-3'): ACGUGACACGUUCGGAGAATT
Cells are seeded in 6-well plates one day before transfection according to the company's guidelines to ensure a cell density of 30%-40% at seeding. siRNA or plasmid transfected by Lipofectamine 3000 reagent (Invitrogen, USA), and subsequent cell function experiments after transfection for 48h. siRNA 1292 was selected as the subsequent lentiviral (Shanghai, Genechem) infection sequence based on the transfection results. After stable transfection 48h, select the target cells with 0.5 µg/mL puromycin for around 14 days.
Quantitative Reverse-Transcription PCR
Extract total cellular RNA using Trizol reagent (Invitrogen) and store at -80°C for later use after determining RNA concentration. According to the Fastking RT Kit (with gDNase) (Tiangen) Usage Guide, synthesize cDNA and store it at -20°C for later use. Using the SYBR Green Assay Mix Kit (Kang Wei Century) on the real-time PCR detection system (Thermo Fisher) perform Real-time PCR experiments. The total working solution system is 20ul, including 0.4ul of forward primer, 0.4ul of reverse primer, 9ul of SYBR reagent, 2ul of cDNA, and 8.6ul of deionized water. The 2−∆∆Ct method determines the relative expression of the target gene. The relative expression of FANCI in each glioma cell was standardized with GAPDH as an internal reference. All experiments were performed in three replicates and were independent.
Primer sequences (AG, Accurate Biology) are as follows:
FANCI: F: 5’-CTCCTCCAAGGGAAGCAGAAAGA-3’
R: 5’-GGCACAGTGACAACATCCAATAGC-3’
CCK-8 Cell Proliferation assay
About 2,000 cells per well were seeded in a 96-well plate, with five replicate wells a day, placed in a cell culture incubator for 1, 2, 3, 4, and 5 days. Each well-added 100µl working solution, which mixed 10µl CCK-8 reagent (Dojindo, Shanghai) and 90µl DMEM, was next put back incubated for 2 hours and then represented proliferative capacity of glioma cells as the absorbance of each well at 450 nm.
EdU Cell Proliferation assay
Place suitable coverslips into 24-well plates, then seed around 8,000 cells per well and incubate for 24–48 hours. Add into per well with 250µl the diluted Edu working solution (20µM) with DMEM, and continue the incubation for two hours. Remove the 24-well plate for 4% paraformaldehyde fixation, and use the EdU cell proliferation kit (Abbkine) to prepare an appropriate amount of staining solution according to the instructions. After staining, BSA is washed twice. Prepare an appropriate amount of 1× Hoechst reaction solution and perform DNA staining for 10 min. BSA wash 2 times and dry moderately. Observe and photograph under an inverted fluorescence microscope and manipulate the relative proliferation rate of cells using ImageJ software.
Wound healing assay
Mark the back of the 6-well plates in advance. Then, inoculate the cells in the 6-well plates at about 1×10^6 cells per well to ensure 100% confluency the next day. Scrape the cell monolayer vertically with a 200µl sterile pipette, wash with PBS, and discard the floating cells. The lower serum DMEM (2%FBS) was substituted for complete DMEM (10%FBS). Remove at 0 and 48h, observe under an inverted microscope, and take pictures. Cell migration area comparison was performed using ImageJ software.
Cell migration and invasion assays
For cell migration, aliquots of 2×10^5 cells with serum-free DMEM per well in the upper chamber of the Transwell insert chambers (pore size 8µm, Corning) without Matrigel, add 600ul of DMEM containing 10% FBS in the lower chamber. For cell invasion experiments, an upper chamber was precoated with Matrigel (Corning). After 48h, the chamber was removed and fixed with 4% paraformaldehyde 30min, stained 10min with crystal violet, and gently wiped the Transwell chamber inner surface with a cotton swab. After properly air-drying, observe it with a microscope and took photography. The number of cells passed through each field of view using ImageJ software processing.
Flow Cytometry
Briefly, take about 1×10^6 cells after cell transfection 48h, with 1800rpm, centrifugation for 5min. cells were resuspended with 400µl of 1× Binding Buffer from apoptosis kit (BD, Biosciences). Then, in the dark conditions add 5µl of propidium iodide (PI) and 5µl Annexin V, mix thoroughly, and incubate at room temperature for 10min. Finally, analyze the apoptosis rate of cells by flow cytometer (Beckman Coulter). Apoptosis rate calculation = (Q1-UR + Q1-LR)/Q1-LL%. Q1-UR represents the proportion of late-stage apoptotic cells in the upper right quadrant, Q1-LR represents the proportion of early-stage apoptotic cells in the lower right quadrant, and Q1-LL represents the proportion of viable cells in the lower left quadrant.
Western blotting
The extraction of total cell protein using RIPA lysate with protease inhibitors and protein quantification by BCA Protein Quantitation Kit (Beyotime). Prepare a 10% protein gel and polyvinylidene difluoride membranes (0.45µm PVDF, Millipore, USA). After the protein is transferred to the membrane, the membrane is closed with 5% skimmed milk powder for two hours, and the primary antibody is incubated overnight at 4°C. The membrane reacted with the corresponding secondary antibody binding reaction at room temperature for 1–2 h the next day, followed by band development using ECL Western Blot luminescence solution (Millipore). In the end, protein band gray value analysis using ImageJ software. The antibodies were as follows: anti-GAPDH (Affinity, 1:1000), anti-FANCI (Abcam, 1:5000), Akt (ABclonal, 1:1000), Phospho-Akt (Ser473) (Always, 1:1000), Bcl-2 (Cell Signaling, 1:1000), Bax (Cell Signaling, 1:1000), HRP-anti-rabbit/mouse (Affinity, 1:5000-1:8000).
Subcutaneous tumor xenografts in nude mice
BALB/c nude mice (4–5 weeks old) were purchased from Peng Yue Animal Breeding Center in Jinan, China. And these mice raised in the SPF-conditioned animal house of Jining Medical College. Stably transfected U251 cells with knockdown for FANCI or negative controls were injected into the right subcutaneous part of each mouse (8 mice in total). After two weeks, observed the growth of the tumor every five days, and sacrificed the mice at four weeks later. Tumors were weighed and treated with 4% paraformaldehyde, and we retained a part of tumor tissue for further histological analysis and RNA extraction. We promise that All experiments involving animals conducted following the protocols of the animal ethics committee approved by the institution.
Fluorescent immunohistochemistry
The tissue sections are first deparaffinized and fixed. The tissue is then repaired with sodium citrate repair solution at high temperatures, and remove the residual solution. Follow the instructions for the fluorescent immunohistochemistry kit (Absin). Block with 5% BSA for one hour at room temperature. Then, incubate with rabbit Ki-67 (Affinity, 1:200) primary antibody for 1 hour at room temperature, and rinse with PBS 3 times for 5 min each time. Dropwise the secondary antibodies, and at room temperature for 1 hour, rinse with PBS 3 times, 5 minutes each time. After DAPI staining, rinse with PBS. Finally, after adding an appropriate amount of autofluorescence quenching reagent to the tissue, the mounting was observed and photographed under a fluorescence microscope after mounting treatment.
Statistical analyses
Data statistical analysis using GraphPad software 8.0. Values from at least three independent experiments. We used the two-tailed unpaired Student’s t-tests to test the statistical differences between the two groups. The One-way ANOVA test for the between-group comparison. The Kaplan-Meier method and log-rank test plotted the survival curve with statistical differences. A p-value less than 0.05 indicates a statistically significant difference.