2.1 Gene expression analysis
We analyzed 33 tumor tissues and their corresponding adjacent normal tissues on Xiantao website (https://www.xiantaozi.com/). To establish non-normal and control groups, we integrated the tumor tissue data from the Cancer Genome Atlas (TCGA) with the normal tissue data from the Genotype-Tissue Expression (GTEx) database for analysis. Besides, we utilized the web-based tool Gene Expression Profiling Interactive Analysis 2 (GEPIA2, http://gepia2.cancer-pku.cn/) to analyze the expressions of MCM5 in multiple tumor types.
2.2 The prognostic analysis
By the "Survival Map" and "Survival Analysis" modules of GEPIA2, the overall survival (OS) and disease-free survival (DFS) maps of MCM5 in gliomas were obtained. Through univariate Cox regression analysis by TCGA database, we analyzed the survival probability of MCM5 and drew a forest plot.
2.3 Clinical factor analysis
The clinical and pathological characteristics of glioma patients were analyzed using relevant information from the TCGA database, and we applied logistic regression to analyze the predictive value of MCM5 expression on the prognosis of glioma patients. Further, the prognosis of MCM5 was verified in detail in each clinical subgroup and survival curves were plotted in Kaplan-Meier analysis.
2.4 The immune subtypes analysis
TISIDB database (http://cis.hku.hk/TISIDB/index.php) is widely used in research related to tumors and immunity. In this study, we studied the relationship between MCM5 expression and the immune of various kinds of cancers by using TISIDB database. Then, the value of MCM5 in each clinical subgroup was verified in detail.
2.5 The functional enrichment analysis
In order to perform functional annotation and Gene Ontology (GO) enrichment analysis on MCM5, the STRING portal (https://string-db.org/) was utilized to search for MCM5 binding proteins and the GEPIA2 "Similar Gene Discovery" module was used to screen out the top 100 target genes related to MCM5 from the TCGA cancer dataset. Besides, Venn diagram cross-analysis was applied to compare the interacting genes of MCM5. In addition, we integrated the above two datasets and performed Kyoto Genome Encyclopedia (KEGG) pathway and GO enrichment analysis.
2.6 Cell culture
Glioma cells (LN18) used in this study were purchased from Shanghai Cell Bank of Chinese Academy of Sciences and cultured in DMEM containing 10% fetal bovine serum. The cells were cultured in a cell incubator with constant temperature containing 5% CO2 at 37°C.
2.6 Cell transfection
Human target gene MCM5 small interfering RNA (siRNA) was provided by GenePharma, with the following sequences: #15'-GCCAAGCUGAAGAAGUUUATT-3', #25'-CCCUCCCAAAUGUCUAUGATT-3', #35'-CGUCCCGGAAUUUCAUCAUTT-3', #45'-AUUUCUGCUUGGUGAAGUCTT-3'. LN18 glioma cells were transfected when the fusion rate reached 60%-70% according to the provided instructions ,using the Lipofectamine 2000 reagent (Invitrogen, Shanghai, China), following the manufacturer’s protocol.
2.7 Cell Counting Kit 8 (CCK-8)
After trypsin digestion and counting, cells were seeded in 96-well plates. At predetermined time points (0h, 24h, 48h, 72h, 96h), we added 10ul CCK-8 reagent to each well and incubate at 37°C for 1 hour, and detected the absorbance of cells at 450nm using an microplate reader and plotted the viability curve to observe the viability of cell proliferation.
2.8 Cell cycle detection
LN18 cells of appropriate density were inoculated in a 6-well plate, and three replicates of siNC, siMCM5-2, and siMCM5-4 were transfected into the cells. After 48 hours, 500,000 cells were resuspended in 1.5 mL of 75% ethanol. Then, cells were fixed overnight at -20°C, centrifuged at 800 rpm for 3 minutes, washed three times with PBS, stained with propidium iodide (PI) for 30 minutes, and then detected by flow cytometry (Syngene; USA).
2.9 Transwell assay
Firstly, the transfected LN18 cells were diluted with serum-free DMEM medium and added to the upper chamber, and DMEM medium containing 10% FBS was added to the lower chamber. After 48 hours of incubation, we removed the small chamber and wiped off excess cells in the upper chamber with a cotton swab. Subsequently, the invading cells were fixed with paraformaldehyde and stained by 0.1% crystal violet. Finally, the number of cell migration was observed through an optical microscope for comparative analysis.
2.10 Wound healing assay
The transfected cells were cultivated to a density of 100%. 200µL microsyringe tip was scratched vertically on a 6-well plate, and then we washed it with PBS to remove exfoliated cells, added 2 ml of medium, and cultured it in a 37°C incubator with 5% CO2. At 0 h, 24 h, 48 h and 72 h, the scratch gap area was observed and photographed under the microscope, and ImageJ was used to analyze the scratch gap area.
2.11 Western blot experiment
Total protein was extracted from LN18 cells after transfection using Ripa lysates. Then, the proteins were separated by SDS-PAGE and transferred to PVDF membrane and sealed in a 5% skimmed milk powder room temperature shaker for 2 h. Afterwards, the primary antibodies targeting MCM5 (cat.11703-1-AP; Protein), CDK (cat.YT5198; Immunoway), CyclinD1 (cat.YT1173; Immunoway), and GAPDH (cat.ET1601-4; Huabio) were incubated overnight at 4°C. The expression levels of MCM5 and cell cycle proteins were detected using ECL chemiluminescence assay kit (cat.P0018AS; Beyotime) after rinsing with TBST buffer.
2.12 Statistical analysis
The measurement data in the online analysis tool are presented as mean ± standard deviation (x ± s). For data that conforms to a normal distribution, t-tests for independent samples are used to evaluate the differences between two groups. For cases with three or more independent samples, one-way analysis of variance (ANOVA) is used to evaluate the differences between groups. All experiments are repeated three times. *P < 0.05, **P < 0.01, ***P < 0.001.