Bioinformatics analysis
The PFKFB4 sequencing expression data and correlation analysis were obtained from TCGA (https://www.cancer.gov/about-nci/organization/ccg/research/structural-genomics/tcga). The differential expression analysis used the "EdgeR" package in R software, and the Pearson correlation analysis was also performed in R. Correlation analysis used the GEPIA database(http://gepia.cancer-pku.cn/. Survival and prognosis analysis of high and low PFKFB4 expression was performed using the OncoLnc database (http://www.oncolnc.org/).
Patients and samples
This study was approved by the Ethics Committee of the Second Affiliated Hospital of Kunming Medical University. Written informed consent from all donors or close relatives was required before using patient samples in this study. Four glioblastomas and adjacent tissues were collected. Patients underwent primary surgery and did not receive radiotherapy or chemotherapy before surgery, and tissues were stored in liquid nitrogen within 2 h of surgical resection.
Cell lines
Human glioma cell lines (U87, U251, and T98G) and human cerebellar astrocytes (HAC) were cultured in Dulbecco's modified eagle medium (DMEM) (Hyclone, Logan, Utah, USA), sh-PFKFB4 U87, and sh-control U87 cells were cultured in MEM (Biosharp, Anhui province, China), 10% fetal bovine serum (Gibco, California, USA), 1% penicillin/streptomycin (Gibco, California, USA) Cells were incubated at 37°C, in a 5% CO2 atmosphere.
Western Blotting (WB)
The antibodies used for western blotting included Anti-PFKFB4 (Abcam,1:3,000, rabbit), Anti-MFN1 (1:1,000; rabbit), Anti-MFN2 (1:1,000; rabbit), Anti-OPA1 (1:1,000; rabbit), anti-β-actin (Cell Signaling, 1:1,000; rabbit), and standard procedures for western blotting. ImageJ (v1.5.2, National Institutes of Health) was used to analyze the bands.
PFKFB4 Interference stabilizes the U87 cell line
The four short hairpins PFKFB4 RNAs (shRNA) were cloned into the lentiviral vector pGMLV-SC5-puro constructed using a high-purity plasmid cassette (DP107, Tiangong, China) and KOD-Plus-Neo (Toyobo, Osaka, Japan). The sequencing results of the PFKFB4 interference vector are shown in Table 1. pGMLV-SC5-puro served as a negative control. The constructed lentivirus vector and auxiliary packaging vector plasmid were co-transfected into 293T cells with the HG transgene reagent (Qiagen, Frankfurt, Germany). The virus-rich supernatant was collected, U87 cells were infected with the packaged lentivirus and the negative lentivirus control, and stable cells were selected with puromycin. The infected cells expressing green fluorescence were detected using flow cytometry, and the infection efficiency was evaluated. qRT-PCR and western blotting were used to further screen for the greatest shRNA3 interference.
Quantitative reverse transcription-polymerase chain reaction (qRT-PCR)
Primers designed by DNAMAN software were synthesized by Sangon Biotech (Table 2). RNAisoPlus (Takara) was added to the cells to extract total RNA. Thermo scientific RevertAid First Strand cDNA Synthesis synthetic cDNA. FastStart Universal SYBR Green Master (Roche)(ROX)amplified. Applied biosystems by Life Technologies To complete the following steps. Three auxiliary wells were used for each sample.
Cell Proliferation Assay
Cell Counting Kit-8 (Beyotime, China) was used to detect cell proliferation. Cells (sh-PFKFB4, sh-control) were seeded in 96-well culture plates (2,500/well, five plates), and the results were recorded for 5 days. One hundred µL of cell culture medium was added as a blank control. absorbed the original medium and adding 100 ul to the ordinary medium containing CCK8 detection solution. After a 2 hour incubation, the absorbance of each well (wavelength 450 nm) was detected by spectrophotometry, and the final value was recorded. The final value = measured absorbance-blank hole absorbance.
EDU
BeyoClick™ EdU-555 (Beyotime, China) was used to detect cell proliferation, and a density of 4 × 105 cells (sh-PFKFB4, sh-control) was inoculated into 6-well culture plates. EdU-labeled cells were fixed with 4% paraformaldehyde, and permeabilized with 1 ml 0.3% Triton X-100 solution, and stained with Hoechst 33342. EdU staining was detected using an inverted fluorescence microscope (20x).
Mito-Tracker Red CMXRos
Mitochondrial labeling was performed using MitoTracker Red CMXRos (Beyotime, China). Cells (sh-PFKFB4, sh-control) were seeded in 6-well culture plates at a 70% cell density, and the MitoTracker working solution was prepared and added to the medium at a ratio of 1: 1,000. The cells were cultured in an incubator for 30 min maintained at 37 °C and 5% CO2, then fixed with 4% paraformaldehyde. The cells were permeabilized, stained with Hoechst 33342, and observed using an inverted fluorescence microscope (20x).
ATP
Collect the cells (sh-PFKFB4, sh-control) in a centrifuge tube and discard the supernatant, add the ATP extraction solution, centrifuge the supernatant to another EP tube after sonication, add chloroform to mix, and then centrifuge to collect the supernatant for testing. The working and standard solutions were prepared according to the kit's instructions. An ultraviolet spectrophotometer was used according to the manufacturer's instructions, and ATP was calculated after the values were measured.
Flow cytometry
Two groups of cells were collected, and the cell density was at least 1x106cells/ml. Annexin Vdome 633 (lot.NY601, DOJINDO, Japan) detected apoptotic cells,) follow the instructions. flow cytometry analyzer (BD FACSCelesta multicolor cell analyzer) test results.FlowJo-V10 analyzed the data.
Immune-deficient mice experiment
This experiment complied with the ARRIVE guidelines, the U.K. Animals (Scientific Procedures) Act, 1986 and associated guidelines, and EU Directive 2010/63/EU. Six immune-deficient male mice (6 weeks old) were purchased from the animal room of Kunming Medical University (Yunnan, China), raised in a room free of specific pathogens (SPF), and randomly assigned to two groups of nude mice, three each. For subcutaneous tumors, 1.2x106 cells (sh-PFKFB4, sh-control) were injected into the left armpit skin (0.2 ml) with an injection time of approximately 1 min. The weights of the mice were recorded weekly. After two weeks, the tumors were dissected, sizes and weights were measured, and western blotting was performed.
Statistics
All the experiments were repeated three times independently. The PRISM 8.0 (GraphPad Software, La Jolla, CA) graphics program and SPSS 21.0 were used for graphical and statistical analysis of the data. The mean ± standard deviation represents the measurement data, and the difference between the two groups was determined using Student's t-test. Statistical significance was set at P < 0.05.