Bioinformatics analysis
|logFC|>1 and P < 0.01 were used as threshold to analyze glioma expression databases GSE12657, GSE35493, GSE104291 and GSE50161 in GEO database (https://www.ncbi.nlm.nih.gov/gds) by R language "limma" package (http://www.bioconductor.org/packages/release/bioc/html/limma.html). Expression database GSE12657 contained a total number of 12 samples, including 5 normal samples and 7 glioma samples. Expression database GSE35493 contained a total number of 19 samples, including 7 normal samples and 12 glioma samples. Expression database GSE104291 contained a total number of 6 samples, including 2 normal samples and 4 glioma samples. Expression database GSE50161 contained a total number of 47 samples, including 13 normal samples and 34 glioma samples. Key genes in these expression databases were obtained by co-expressed analysis through “Robust Rank Aggreg” (https://cran.r-project.org/web/packages/RobustRankAggreg/index.html). HTF target (http://bioinfo.life.hust.edu.cn/hTFtarget#!/) and Cist Rome (http://cistrome.org/) were used to obtain the names of human transcription factors. The key transcription factors were obtained by comparing the names of key genes and transcription factors, taking the intersection and combing with the existing literature. The possible downstream regulatory pathways were predicted through the existing literature, and the relationship of the downstream pathways was verified by correlation analysis and MEM (https://biit.cs.ut.ee/mem/index.cgi) co-expression analysis.
Sample collection
Sixty of glioma specimens by surgical removal which were confirmed by pathology analysis were collected from January 2014 to January 2015. These selected patients (male, 38, female, 22; aged range 49–71, the average age 61; stageⅠ-Ⅱ, 31, stage Ⅲ-Ⅳ, 29; KPS score > 70, 35, KPS score < 70, 25) were the first time for surgical treatment. Excluded criteria: patients with other malignant tumor concurrent, incomplete clinical information, serious heart disease, kidney disease and lung dysfunction; In addition, 24 cases of normal brain tissues removed by internal decompression surgery due to severe craniocerebral injury were taken as the control group, all of which were not treated with chemoradiotherapy before surgery. Sixty glioma patients were followed up until January 2020 by telephone or follow-up visit. The 3-year OS of each patient was observed. By the end of follow-up, 2 out of 60 patients had lost to follow-up, with a follow-up rate of 96.67%. Follow-up time was 5–36 months. All patients in this study signed the informed consent and were approved by our medical ethics committee to comply with the Helsinki declaration.
Cell culture and transfection
Glioma cell lines A172 and U251 cells provided by Stem Cell Bank, Chinese Academy of Sciences were cultured in 1640 medium containing 10% fetal bovine serum, 100 U/mL penicillin and 100 mg/mL streptomycin at 37℃ in 5% CO2.
According to the experimental requirements, cells were transfected respectively. When the cell density reached 90% and was in the logarithmic growth phase, cells were digested with trypsin, made into cell suspension (2.5 × 104 cell/mL), and inoculated on 6-well plates (2 mL for each well). Lentiviral vectors were constructed using lv5-gfp (lentiviral gene overexpression Vector), psih1-h1-copgfp siRNA Vector (lentiviral siRNA fluorescence expression Vector gene silencing Vector). Si-ELF1, si-GFI1, oe-MEIS1 and their negative controls were all constructed by Thermo Fisher Scientific (Waltham, MA, USA). Lentivirus was packaged in 293T cells, which were cultured in RPMI-1640 complete medium containing 10% fetal bovine serum and passed every other day. When the A172 and U251 cells were in the logarithmic growth phase, they were digested by trypsin and blown, and 2 mL cell suspensions (1 × 105 cells/mL) were inoculated on 6-well plates, and cultured overnight at 37℃. Then the virus (1 × 108 TU/ml) was added to the cells for infection, and cells with stable heredity were obtained and collected for subsequent experiments. Sequence of si-ELF1: GGATGTTGCTGAAGAAGAA, Sequence of si-gif1: CGAGCAGACAGCACTTCAA.
RT-qPCR
Total RNA (Invitrogen, Car, USA) was extracted according to the instructions of Trizol method, and the RNA was reversely transcribed into cDNA using Prime Script RT kit (RR037A, Takara, Shiga, Japan) with a system of 10 µL. Then the reaction liquid was exposed to fluorescent quantitative PCR based on operate instruction of SYBR® Premix Ex Tag™ Ⅱ kit (RR820A, Takara). The samples were subjected to real-time quantitative PCR using a real-time quantitative PCR system (ABI 7500, ABI, Foster City, CA, USA). With GAPDH as internal control, the 2 - ΔΔ Ct method was used to calculate the relative gene expression. Relevant primers were assigned to Shanghai Sangon Biotech (Shanghai, China) (Table 1).
Protein extraction and quantification
About 1 × 106 cells were treated with 1 ml cell lysate (containing protease inhibitor) (P0013J, Bey time biotechnology CO., LTD., Shanghai, China) for 45 min, and then cleaved for 30 min at 4℃ and 8000 rpm to collect the supernatant. And the protein concentration of each sample was determined using the BCA kit (PC0020, Beijing Solebar biotechnology CO., LTD., Beijing, China). After electrophoresis, the proteins were transferred to a nitrocellulose membrane (66485, PALL, NY, USA). After membrane transformation, the membrane was blocked in 5% skim milk at room temperature for 2 h, and washed with trimethyl aminomethane buffer brine (TBS/T) for 3 times, each time for 10 min. The membrane was incubated with the primary antibodies ELF1 (ab64937, 1:500, Abcam, USA), MEIS1 (ab19867, 1:1000, Abcam, USA), GFI1 (ab21061, 1:1000, Abcam, USA), MMP2 (ab92536, 1:1000, Abcam, USAUSA), PCNA (ab92552, 1:1000, Abcam, USA), Caspase 3 (ab13847, 1:500, Abcam, USA), cleaved-caspase 3 (ab32042, 1:500, Abcam, USA) and GAPDH (ab9485, 1:1000, Abcam, USA). The next day, the membrane was washed at room temperature with TBS/T for 3 times, each time for 5 min. Horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (ab97051, 1:2000, Abcam, USA) was added and incubated at room temperature for 1 h. The membrane was washed with TBS/T for 10 min × 3 times, immersed in ECL reaction solution (BM101, Bioimage, USA) for 1 min, and then exposed with X-ray in the dark, and finally the target protein bands were measured. GAPDH was used as internal parameter, and the ratio of gray value of target band and internal reference band was used as the relative expression of protein.
Cell proliferation assay
After transfection, A172 and U251 cells were digested and resuscitated. The cell concentration was adjusted to 1 × 105 cells/mL, and the cells were inoculated into a 96-well plate with 100 µL/well and routinely cultured overnight. Cells were treated according to the instructions of CCK-8 kit (Beyotime, Shanghai, China), and cell viability was measured by CCK-8 at 24, 48, 72, and 96 h after inoculation. At each test, 10 µL of CCK-8 detection solution was added, incubated in the incubator for 4 h, and absorbance at 450 nm was tested with an enzyme marker, and the growth curve was drawn.
Cell invasion ability experiment
In the transwell invasion experiment, the Matrigel stored at -80℃ was melted into a liquid overnight at 4℃. And 200 µL of Matrigel was added to 200 µL of serum-free medium. And then 50 µL of Matrigel were added to upper chamber and incubated for 2–3 h until the gel became solid. Cells were digested and counted, and cell suspension was prepared with serum-free medium. Next, 200 µL cell suspension was added to the upper chamber of each well, and 800 µL cell suspension was added to the lower chamber containing 20% FBS conditioned medium. Cells were incubated at 37℃ for 20–24 h. After that, transwell plate was soaked in formaldehyde for 10 min, and rinsed with pure water for 3 times. The cells were stained with 0.1% crystal violet at room temperature for 30 min, and the cells on the upper surface were wiped off with cotton balls. Cells on the membrane were observed, photographed and counted by an inverted microscope. Substrate glue was not required for transwell migration experiment, and the incubation time was 16 h. Cells from at least four randomly selected microscope regions were counted.
Cell apoptosis assay
After transfection for 48 h, the cells were digested with 0.25% trypsin and collected in the flow tube, centrifuged, and the supernatant was discarded. Annexin-V-FITC, PI, and HEPES buffer were incorporated into annexin-V-FITC/PI dye at a ratio of 1:2:50 according to the instructions of Annexin-V-FITC apoptosis assay kit (559763, Becton, Dickinson and Company, NY, USA). And 1 × 106 cells were resuspended in 100 µL of dye solution, and the cells were oscillated and mixed. After incubation at room temperature for 15 min, 1 mL HEPES buffer (PB180325, Porcello, Wuhan, China) was added to the solution for oscillating and mixing. FITC and PI fluorescence were detected by excitation of 525 nm and 620 nm bandpass filters at the wavelength of 488 nm to detect cell apoptosis.
Chromatin immuno-coprecipitation (ChIP)
The EZ-Magna ChIP kit (EMD Millipore) was used for ChIP determination. According to the manufacturer's protocol, the cells were immobilized with 4% paraformaldehyde and incubated with glycine for 10 min to produce DNA-protein cross-linking. The cells were then lysed with a cell lysis buffer and a nuclear lysis buffer and treated with ultrasound to produce 200–300 bp of chromatin fragments (a portion of the DNA as INPUT). Next, lysates were immunoprecipitated by magnetic protein A beads antibodies bonded with various antibodies. H3K27ac antibody (ab177178, Abcam, USA) or H3K4me1 (ab176877, Abcam, USA) were added to the target protein group. Negative control was added with rabbit IgG (ab171870, Abcam, USA; RRID). Finally, the precipitated DNA was analyzed by RT-qPCR.
In vivo animal experiment
Ten BALB/c male nude mice (age: 4–5 weeks old; weight: 18–22 g) were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China). Si-ELF1 was used to construct lentivirus vector to obtain stable interference expression of ELF1 and negative control of human U251 cell line. Cell suspension (20 µL, 1.0 × 106 cells/mL) was inoculated in nude mice at abdominal subcutaneous. Tumors were observed weekly and measured with vernier caliper. Calculation formula of tumor volume (TV) is: TV = 1/2 *a*b2, where a is length of tumor and b is width of tumor. Mice were exposed to euthanasia at 35 d, and tumors of each groups were removed, weighted and photographed. All the above experimental animals have been approved by the animal protection and use committee, and all the animal experiments in this study are in accordance with the management and use principles of local experimental animals.
Immunohistochemistry
Paraffin-embedded tumor tissues were subjected to dewaxing, hydration, xylene I, II dewaxing, and gradient alcohol dehydration. Sections were immersed in 3% H2O2 for 10 min, washed with PBS twice for 5 min and repaired with antigen (Beyotime, Shanghai, China) at high pressure for 90 s, and then cooled down at room temperature. Sections were blocked with 5% BSA at 37℃ for 30 min, and incubated with primary rabbit antibody at 4℃ overnight. Then tissues were incubated with HRP labeled goat anti rabbit (ab205718, 1: 1000Abcam) at 37℃ for 30 min. Finally, sections were exposed to DBA solution (MXB, Fuzhou, China), stained with hematoxylin for 5 min, and observed by optical microscope (XSP-36, BSD., Shenzhen, China) and photographed. Five high-power fields were randomly selected from each section, and 200 cells were counted in each field. The number of positive cells < 5% was negative, and the number of positive cells ≥ 5% was positive. The immunohistochemical results were scored by two people independently using double-blinded fashion.
Statistical analysis
All the present data were expressed as mean ± standard deviation of three independent experiments. The significant difference from the respective control for each experimental test was assessed by one-way analysis of variance (ANOVA) using SPSS21.0 software (IBM Corp, Armonk, NY, USA). The difference is considered significant if p < 0.05.