Cell Lines
Glioma cells (U87, U251, T98, U118) were procured from the Procell (Wuhan, China) and cells were grown in Dulbecco's modified Eagle's medium (DMEM) (Gibco Company, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS) (FBS, Gibco Company, Grand Island, NY, USA). Normal human astrocytes (NHAs) grown in astrocyte media (AM, ScienCell, USA, Cat. No. 1801) were provided by Sciencell Research Laboratories. Cell lines were cultivated at 37C with 5% CO2 in a humid incubator.
Microarray data analysis
The GSE25631 dataset including data from the sequencing process from GEO database. The GSE25631 dataset includes 82 primary glioblastoma multiforme surgical specimens and 5 normal brain tissues from locations around arteriovenous malformations.
Quantitative real‑time polymerase chain reaction (qRT‑PCR)
The RNA isolater (Vazyme, Nanjing, China) was used to extract total RNA from glioma tissues and cell lines, as directed by the manufacturer. According to the manufacturer's instructions, cDNA was produced using a miRNA 1st Strand cDNA Synthesis Kit (by stem-loop) (Vazyme, Nanjing, China). Vazyme created stem loop miRNA qRT-PCR primers specific for miR-760 and U6. (Generay, Shanghai, China). The primers mentioned in this paper are shown in Table 1. The CT results were analyzed by the 2-ΔΔCT method.
Cell Transfection
miR-760 or NC mimics, shRNA for MMP2 knockdown (sh-MMP2) and negative control (sh-NC), overexpression of MMP2, and the control group were manufactured at GenePharma Company (Shanghai, China). The sequences mentioned in this article are shown in Table 2. According to the manufacturer's recommendations, Lipofectamine 3000 (Invitrogen) was utilized to transfect cells. The relative expression of miR-760 and MMP2 was determined 48 hours later using a qRT-PCR experiment.
Dual-luciferase reporter assay
The wild-type (WT) and mutant-type (MUT) 3′UTR (non - translated region) sequences of MMP2 including the miR-760 binding site were initially produced. These sequences were created by Genechem (Genechem Co.,Ltd. Shanghai, China). miR-760 mimics and inhibitors were transfected into glioma cells using Lipofectamine 3000, as well as MMP2-WT or MMP2-MUT. The cells were then grown for further 48 hours before being harvested. The Dual-Luciferase Reporter Assay System was then used to calculate the luciferase activity (Promega, USA). Experiments were conducted out according to the producers' directions.
EDU assay
The KeyFluor555 Click-iT EdU Kit (Keygen, Jiangsu, China) were employed for the 5-ethynyl-2'-deoxyuridine (EdU) test. Transfected U87 and U251 cells were planted at a density of 2×104 cells per well in 24-well culture plates and cultivate for 12 hours. After a further 12 hours of incubation, PBS was used to rinse the treated cells twice. The cells were fixed with 4% paraformaldehyde for minutes at 37 °C after being incubated with 10μM EDU solution at 37°C for 2 hours. For 30 minutes, the nuclei of the cells were dyed with DAPI. Lastly, using a fluorescent microscope(Carl Zeiss, Germany), the EdU-positive cells was shown and assessed. The proliferation ratio was determined by counting EdU positive cells then dividing the quantity of EdU positive cells by the total amount of cells in ratio.
Transwell assay
U87 and U251 cells were transplanted at 37°C for 24 hours in the top chamber with or without Matrigel after transfection pretreatment (BD Biosciences). After 48 hours, cells migrated to the membrane's bottom were dyed with Crystal violet (Beyotime), and cells left on the membrane's top surface were cleaned using a cotton swab.
CCK-8 assay
96-well plates with 2000 cells per well were seeded and cultivated. At 0, 24, 48, and 72 hours, CCK8 working solutions were added and the culture continued for an extra 2 hours. The wavelength of the plate was determined for each 450nm time point.
Western Blot
To extract total protein, cells were lysed according to the manufacturer's instructions using the radio-immunoprecipitation assay (RIPA) lysis buffer (YIFEIXUE BIO TECH, Nanjing, China). SDS-PAGE was used to isolate proteins and then transfer them to polyvinylidene difluoride (PVDF) membranes (Millipore Corporation, Billerica, USA). After one hour of blocking with 5% skim milk, the membranes were incubated at 4 °C for 12 hours with primary antibodies (anti-MMP2 and anti-β-actin). Then, a secondary antibody conjugated to HRP was added and incubated for an additional 1 hour. To measure protein expression, an enhanced chemiluminescent system (Biosharp, Hefei, China) was utilized. In this experiment, β-actin was employed as a referent gene.
Immunofluorescence staining
U87 and U251 cells were grown on glass coverslips until they were 50 percent confluent, then fixed for 30 minutes with 4% paraformaldehyde. After that, the cells were blocked with donkey serum for minutes at 37 °C, then coated overnight at 4 °C with anti-MMP2 antibody (1:50, MMP2,proteintech, China), followed by 1 hour at 37 °C with goat anti-rabbit IgG (1:200, proteintech, China). The nuclei were then dyed for 5 minutes with 4′,6-diamidino-2-phenylindole (DAPI; Beyotime, China). Finally, the cells were examined using fluorescence microscopy.
Statistical Analysis
SPSS 20.0 (IBM Corp, Armonk, NY, USA) was used to process the data. The data are then given as the mean±SD of three separate experiments. Analysis of variances were applied using the student t test and one-way ANOVA, P<0.05 was deemed significant statistically.