2 − 1 Data processing
We downloaded clinical information of patients with glioma mRNA expression patterns from The Cancer Genome Atlas (TCGA, https://portal.gdc.cancer.gov/repository), including five cases of normal samples and 698 cases of tumor samples.
2–2 Statistical analysis
The Wilcoxon signed-rank test and logistic regression were used to evaluate the relationship between clinical factors and MTMR14 expression. Cox regression and Kaplan–Meier methods were used to determine the clinical factors associated with overall survival in patients with glioma. Multivariate Cox regression analysis was used to investigate the role of MTMR14 expression in survival and other clinical characteristics (age and stage). High and low expression of MTMR14 were determined according to the median value, based on which all patients were divided into low-and high-expression groups. All statistical analyses were performed using R software (version 4.1.1).
2–3 Functional enrichment analysis
Gene Set Enrichment Analysis is a calculation used to determine whether a predefined set of genes exhibits significant differences between two biological states[23, 24]. To investigate the potential mechanism by which MTMR14 expression affects LGG progression, we used R software (V.4.1.1) to screen for biological pathways with significant differences between the MTMR14-high and MTMR14-low groups. For each analysis, the genome was sequenced 5,000 times. Genomes with a false discovery rate < 0.05 and adjusted P value < 0.05, were considered significantly enriched, and significantly enriched gene ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were identified.
2–4 Construction of protein-protein interaction (PPI) network and selection of hub genes
MTMR14 was imported into the STRING database (version 11.5B, https://string-db.org) to identify physically and functionally interacting genes and proteins. The PPI network was constructed with a comprehensive score > 0.4 and further visualized using Cytoscape version 3.9.1. The CytoHubba plugin selected (http://www.cytoscape.org/) the neutral (MCC) algorithm through the largest group PPI network degree of interaction in the top five hub genes[26, 27].
2–5 Immune cell infiltration
The expressions of MTMR14 and myeloid dendritic cells (DCs), CD + T cells, macrophages, and neutrophils were analyzed with the TIMER2.0 (http://timer.cistrome.org/) database. The threshold for MTMR14 was significantly associated with immune cell infiltration (P < 0.001).
2–6 Reagents and Antibodies
Culture medium (DMEM), fetal bovine serum (FBS), Cell Counting Kit (CCK8) kit, and a cell cycle and apoptosis detection kit were purchased from Servicebio (Wuhan, China). Akt, P-Akt, Bax, P-mTOR, caspase-3, caspase-9,Bcl-2, and β-actin antibodies were purchased from Servicebio (Wuhan, China) .
2–7 Cell culture
Glioma cell lines (U87 and U251) were purchased from the Chinese Academy of Medical Sciences (Beijing, China) and cultured in DMEM containing 10% FBS in an incubator at 5% CO2 and 37°C.
To verify and screen for better interference fragments, cells were divided into a group with interference fragment 1 (S1), a group with interference fragment 2 (S2), and a group with no load (NC). The interference fragment and riboFECTTMCP were mixed at a ratio of 1:2 and inoculated at a concentration of 50 nm on a six-well plate with a growth density of approximately 50%. The interference efficiency was analyzed using real-time polymerase chain reaction (PCR) after 24 h of interference.
2–9 Real-time fluorescence quantitative nucleic acid amplification detection experiment
Total RNA was isolated using TRIzol (Servicebio) and then reverse-transcribed into complementary DNA using the Green qPCR Master Mix Kit (Servicebio). The primer sequences are listed in Supplementary Table S1. A two-step quantitative RT-PCR (Servicebio) was used for quantitative PCR. To verify the efficiency of the interference fragment, an interference fragment with a better interference effect was selected for subsequent experiments.
2–10 Experimental grouping
The cells were divided into a control group without any treatment, an S group with interfering fragment, and an NC group without interfering fragment vector.
2–11 Cell viability assay
Cells (2 × 103) were cultured evenly on 96-well plates for 2, 24, and 48 h, after which 10 µL CCK8 reagent was added and cultured at 37°C for 30 min. The absorbance of the solution was measured at 490 nm using a microplate reader.
2–12 Wound healing assay
U87 and U251 cells were cultured in 6-well plates. When they reached approximately 85% confluence, they were scraped with a new 200 µL pipette tip and washed twice with cold phosphate buffer solution (PBS). Photographs were taken at 0, 24, and 48 h for the S, NC, and CON groups, respectively. The ImageJ software was used to collect images and quantify the gap distance.
2–13 Flow cytometry apoptosis assay
ServiceBio cell cycle and apoptosis assay kits were used. Glioma cells were inoculated in 6-well plates, and the S, NC, and CON groups were treated for 48 h. Cells were collected after trypsin digestion, washed twice with PBS, centrifuged for 5 min (1000 × g), and the supernatant was collected. Centrifugation was conducted at 5–10 × 104 cells for 5 min (1000 × g) and suspended in 480 µL of combined buffer. Add RNase A (10 µL) and Propidium Iodide (PI, 10 µL) were added and incubated at 37°C in the dark for 30–60 min. Fluorescence was detected and analyzed using flow cytometry at an excitation wavelength of 488 nm.
2–14 Immunofluorescence assay
The cells were cultured in a 12-well plate, transfected 24 h later, and fixed in a 12-well plate, after which properly diluted fluorescent-labeled antibody solution was added to completely cover the specimen, and they were incubated overnight at 4°C. The following day, excess water was absorbed with filter paper, and the specimens were immediately observed under a fluorescence microscope (Leica China).
2–15 Western blotting analysis
U87 and U251 cells were cultured and treated in 6-well plates. They were then cut in an ice bath in radioimmunoprecipitation assay buffer for 30 min and centrifuged at 4°C and 12,000 × g for 15 min. The extracted supernatant was stored at -80°C for analysis. Protein concentration was determined using the BCA method (Beyotime). Equal quantities of proteins were loaded onto a 10% sodium dodecyl sulfate-polyacrylamide gel for electrophoresis and then transferred by electrophoresis to a polyvinylidene fluoride membrane (Millipore, Boston, MA, USA). For blocking, 5% bovine serum albumin was used at room temperature for 2 h, and the primary antibody was incubated overnight at 4°C. Tris buffer brine and Tween-20 washing solution were mixed three times (5 min/time), dual fluorescence antibody at 1:20,000 dilution was added, and the broth was incubated at room temperature for 1 h. SPECTRA MAX SoftMax Pro analysis software was used to detect the optical density (OD) signal intensity of each band on the film. β-actin was used as a loading control and for standardization.
2–16 Statistical analysis
All data are expressed as mean standard deviation (SD). The experiments were repeated at least three times, and two groups were compared using an independent t-test. Univariate ANOVA was used among multiple groups, and GraphPad Prism8 software was used for comparative analysis. P < 0.05 was considered statistically significant.