Bioinformatics analysis of FAM84B expression and mechanism

TCGA GBM RNA-Seq data were downloaded from https://xenabrowser.net using the UCSC Xena Browser. The differences in gene expression of FAM84B in glioma and control groups were analyzed, and the ROC curves of FAM84B gene expression were plotted using the R package “pROC.” In addition, the CPTAC module in the UALCAN database (http://ualcan.path.uab.edu/) was used to view the difference in protein levels of FAM84B between glioma and control groups. The level of immune invasion in gliomas was calculated using the ssGSEA method and the correlation of FAM84B gene expression with 28 immune cells was analyzed.

Select the Gene Analysis Module in the TISCH database (http://tisch.comp-genomics.org), enter FAM84B, and select Glioma to view FAM84B expression in various cell types. In addition, select the “Glioma_GSE89567” dataset in the data module to view the cell distribution, and enter the gene set of KEGG’s cell cycle pathway to view the difference in cell cycle pathway fraction in different cells.

The TCGA GBM RNA-Seq data was used to perform a Bayesian t-test by dividing the FAM84B median expression into high and low expression groups by the R package “limma.” KEGG pathway enrichment analysis was performed based on the R package “GSEA,” and values with P less than 0.05 were considered statistically significant. The cell cycle pathway score in glioma was calculated using the ssGSEA method, and the correlation between FAM84B gene expression and cell cycle pathway score was analyzed.

Main reagent materials

Glioma cells, U251, T98, LN-229, and U87, were purchased from the Cell Bank of the Chinese Academy of Sciences; cell culture medium, trypsin, and FBS were purchased from Zhejiang Tianhang Biotechnology Co., Ltd.; penicillin/streptomycin was purchased from Sigma, USA; TRIzol reagent was purchased from Invitrogen Reagent, USA; AMV one-step RT-PCR kit and FAM84B and β-Actin primers were purchased from Sangon Biotech (Shanghai) Co., Ltd.; FAM84B siRNA was purchased from Guangzhou Ruibo Reagent Company; MTT reagent, cell cycle detection kit, and dual-luciferase assay kit were purchased from Shanghai Beyotime Biotechnology Inc.; ELISA apoptosis detection kits were purchased from Roche Reagent, Switzerland; protein lysate was purchased from Beijing Solarbao Science & Technology Co., Ltd.; BCA protein concentration detection reagent was purchased from Shanghai Biotrive Sci&Tech. Co., Ltd.; ECL chemiluminescence kits were purchased from Thermo, USA; Wnt3a (ab219412), β-catenin (ab68183), cMYC (ab32072), Cyclin D1 (ab16663), and GAPDH (ab9485) antibodies were purchased from Abcam, UK.

Clinical samples

Blood samples from 40 patients with glioma admitted to our hospital from January 2013 to December 2019 were selected according to the inclusion criteria, as follows: patients who did not undergo radiotherapies, and chemotherapies; patients with glioma confirmed by pathology; patients without other organ tumors; and patients with informed consent signed by themselves or their family members. The mean age of included patients was 61.36±9.25 years old. Another 20 blood samples of healthy controls were collected as normal controls, with patients’ average age in the control group of 55.27±10.88 years old. Ethics has been approved by the Ethics Committee of our hospital.

Cell culture

Glioma cells, U251, T98, LN-229, and U87, were all cultured in the culture medium containing 10% FBS and 100U/ml of penicillin/streptomycin, which were placed in a incubator with 5% CO2 at 37°C.

qRT-PCR

Patients’ plasma and cells were collected and cells were lysed by adding the TRIzol reagent. After the tissues and cells were completely dissolved, chloroform solution was mixed evenly, the upper transparent liquid was sucked into the EP tube containing isopropanolat after the low-temperature and high-speed centrifugation, in which RNA precipitation was obtained. After being washed with ethanol absolute twice, RNA was dissolved by adding double distilled water without RNA enzyme. The AMV one-step RT-PCR kit was adopted for RNA reverse transcription and PCR amplification, with 1μl of 10X one-step RT-PCR buffer, 1μg of RNA and 0.2μl of each upstream and downstream primer, 0.1μl of AMV RT, 0.1μl of RNase inhibitor, 1μl of Taq DNA polymerase, and 10μl of double steamed water without RNA enzyme for supplement. After operating, the reverse transcription was performed at 45°C for 20min, pre-denaturation at 94°C for 5min, denaturation at 94°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s, for a total of 40 cycles, as well as the final extension at 72°C for 10min. According to the internal reference gene GAPDH, the data were normalized for each parallel sample, and the relative FAM84B expression was calculated with 2–∆∆Ct. All the experiments were repeated three times. The primer sequences of FAM84B were F: 5′-GACCCACCTAAGTTACAAGGAAG-3′, R: 5′-GTAGAACACGGAGCATTCCAC-3′. Those of β-Actin were F: 5′-AGAAGGCTGGGGCTCATTTG-3′, R: 5′-AGGGGCCCACAGTCTTC-3′.

Cell transfection

Glioma cells with the highest FAM84B expression were selected for FAM84B siRNA transfection. The cells in the log phase were collected with tryptase digestion and were inoculated into 6-well plates at 2×105/well, which were divided into si-NC and si-FAM84B groups. After the incubation for 12h in the incubator, siRNA of each group was mixed with lip 2000 and the medium, which was added to the cells. After the culture for 6h, the medium was replaced for fresh, followed by another 48h of culture. The sequence of si-FAM84B-1 was 5′-GCAACCAGGTGGAGAAATT-3′ and of si-FAM84B-2, 5′-GGAATAGAATCATAGTTAA-3′. FAM84B expression was detected by qRT-PCR, to verify the effect of FAM84B siRNA transfection.

Cell proliferation detection for MTT assay

The cells were collected by trypsin digestion and inoculated in 96-well plates at 1500 cells/well, with 150μl of the medium. Each group has 6 parallel samples, which were cultured in the incubator. A total of 20μl of the MTT reagent was added to each sample cell medium at 0h, 24h, 48h, and 72h of cell adhesion and mixed by gently shaking. After the incubation for 4h in the incubator, the cell culture medium was discarded, with 150μl of DMSO added, accompanied by gentle shaking for mixing. After the complete dissolution of crystals, the cell absorbance (OD) of samples at 490nm was analyzed with the full wavelength scanner.

Flow cytometry detection for cell cycle

The cells were collected by trypsin digestion, resuspended in cold PBS, and fixed in 75% ethanol overnight. Fixed cells were washed with PBS for three times and collected by centrifugation, which were incubated with the buffer containing 50 g/ml propidium iodide (PI) and RNA enzyme at 37°C for 1h. Cell cycle distribution in each group was analyzed by flow cytometry detection.

Western blotting

The cell plaques in each group were collected by tryptic digestion and washed in PBS for three times. Protein lysate was added to completely lyse the cells on ice, which were centrifuged at 4°C and 14000g at ultra high-speed for 30min. The centrifuged cell fragments were discarded and the protein concentration was detected with a BCA assay kit. The protein supernatant was added to the protein loading buffer and boiled in boiling water for 3min. A total of 20μg of protein sample loading was taken for gel electrophoresis to separate the target proteins. The protein was transferred to the PVDF membrane, on which the non-specific protein was sealed with the skimmed milk. After washing with TBST for three times, the PVDF membrane was incubated with target primary antibody dilution (Cyclin D1 1:1000, CDK2 1:1000, CDK4 1:1000, CDK6 1:1000, P53 1:500, P21 1:500, and GAPDH 1:1000) for 4°C, which was incubated with secondary antibody dilution (1:8000) at room temperature for 1h after washing with TBST for more three times. After washing with TBST for another three times, protein bands were exposed with the ECL kit. Images were taken with the BIORAD gel imaging system, which were processed with Image Lab3.0 software. The gray value of each protein footprint was obtained, and GAPDH was used as a reference to quantitatively analyze the gray ratio of the target protein to the internal reference in each treatment group.