2.1 Cell Culture
Human glioma cells U87 MG, U251, U118 MG, A172, T98G, LN18, LN229, and human umbilical vein endothelial cell (HUVEC) were purchased from the American type culture collection (ATCC). All cell lines were cultured in DMEM (Gibco, CA, USA) supplemented with 10% fetal bovine serum (FBS, Sigma-Aldrich, MO, USA) and 1% penicillin/streptomycin (Gibco, CA, USA), under 5% CO2 atmosphere at 37°C.
2.2 Cell Proliferation Assay
Cell proliferation was determined by a cell counting kit-8 (CCK8; Beyotime, Shanghai, China). Cells were seeded in 96-well plates at a density of 5 × 103 cells per well overnight before the application of different treatments. After 48 h, 10 µl CCK8 reagent was added to each well, and the plates were incubated for 2 h at 37°C. The optical density (OD) of each well was measured at 450 nm.
2.3 Wound Healing Assay
U87 MG and T98G cells were seeded in 6-well plates. The cells were scratched with a 1-mL pipette tip when the cells reached 90% confluence, and floating cells were washed twice with phosphate saline (PBS). The cells were then incubated in DMEM containing 3% FBS. The wound width was measured at different times by a microscope and the rate of wound closure was calculated.
2.4 Cell Invasion Assays
The invasive capacity of cells was tested by 24-well Matrigel invasion chambers (Corning, NY, USA) with an 8-µm pore membrane. U87 MG and T98G cells were seeded in the upper chamber of a transwell device with 0.2 mg/ml matrigel (Corning, NY, USA) at a density of 2 × 104 cells per well in 200 µl serum-free medium, and 700 µl complete medium was added to the lower chamber. After 24 h, the medium was removed from the upper and lower chambers, the cells were fixed with 4% paraformaldehyde for 20 min and then stained with 0.1% crystal violet for 15 min. Cells that failed to penetrate the surface of the upper chamber were carefully wiped off with a cotton swab, and the penetrated cells were counted under a microscope. Five fields per chamber were randomly counted for statistical analysis in triplicate.
2.5 siRNA Synthesis and Transfection
Small interfering RNA against COL11A1 and the negative control siRNA were chemically synthesized by Transheep (Shanghai, China). The sequences of the COL11A1 siRNA were as follows: 5’-GCAUGGUAUUCAGCAAAUUdTdT-3’ (sense) and 5’-AAUUUGCUGAAUACCAUGCdTdT-3’ (antisense). Lipofectamine 3000 transfection reagent (Invitrogen, Carlsbad, CA, USA) was used for siRNA transient transfection, according to the manufacturer’s instructions. The cells were collected and subjected to subsequent analysis 72 h after transfection.
2.6 Western Blot
After treatment with berberine (Chroma, Chengdu, China) or TGF-β1 (Peprotech, NJ, USA), the cells were lysed with an RIPA lysis buffer (Beyotime, Shanghai, China) with added protease inhibitor (TargetMol, Shanghai, China), and an equal amount of protein was separated by the SDS-PAGE gel. Primary antibodies anti-COL11A1, anti-MGMT, anti-MMP2, anti-MMP3, anti-MMP9, and anti-TGF-β1 were purchased from Abcam (Cambridge, UK); anti-GAPDH antibodies were purchased from Engibody (DE, USA).
2.7 Enzyme Linked Immunosorbent Assay
Human TGF-β1 enzyme linked immunosorbent assay kit was purchased from Cusabio (Wuhan, China). U87MG and T98G cells were treated with berberine for 48 h and then centrifuged at 4°C 1000 × g for 15 min to obtain the supernatant. According to the manufacturer's instructions, samples were acidified with 1 N HCl for 10 min and neutralized with 1.2 N NaOH to activate TGF-β1 in the samples. Biotin-labeled antibody working solution and horseradish peroxidase-labeled avidin working solution were sequentially added into an enzyme-labeled plate; finally, a substrate solution and a termination solution were added, and the OD values of each well were measured by a microplate reader at a wavelength of 450 nm within 5 min.
2.8 Xenograft Experiments
All animal experimental operations were performed in accordance with the procedures for experimental animals, approved by the Animal Ethics Committee of the First Hospital of Jilin University (No. 20210547). BALB/c-nu male mice aged 5-6 weeks were purchased from Charles River Laboratories (Beijing, China), and 2 × 106 U87 MG cells were implanted subcutaneously. Five mice were used in each group. The mice were intraperitoneally injected with 5 mg/kg berberine and the same volume of solvent, respectively. Tumor length and width were measured with vernier caliper every 3 days. Tumor volume was calculated according to the following formula: volume = 1 / 2 × length × width2. Two weeks after the berberine treatment, the mice were sacrificed by cervical dislocation after intraperitoneal injection of pentobarbital sodium (100mg/kg) and the tumor tissue was excised.
2.9 RNA-Sequencing Analysis
According to the manufacturer’s protocol, the isolated nude mice xenografts were placed on ice and the total RNA was extracted with RNAeasy Animal RNA Isolation Kit (Beyotime, Shanghai, China). For high-throughput sequencing, the libraries were prepared, following the manufacturer's instructions, and applied to Illunima HiSeq X Ten system for 150 nt paired-end sequencing. We used edgeR software, which is dedicated to performing differential gene expression analysis. The mathematical model used was a negative binomial distribution model. EdgeR allows analysis of the differential expression between two or more samples, and the analysis results used fold change (FC) and false discovery rate (FDR) values to determine whether a gene was differentially expressed. Significant expression difference was defined as FC of ≥ 2 or ≤ 0.5 and FDR of < 0.05.
Paraffin sections were dewaxed, dehydrated with gradient alcohol, subjected to antigen repair, and then rinsed with 0.01 M PBST for 5 min × 3 times. In addition, 3% BSA was blocked in a wet box for 30 min. Anti-COL11A1 (abcam, Cambridge, UK) was incubated overnight at 4°C and rinsed with 0.01 M PBST for 5 min × 3 times. The sections were incubated with CY 3-labeled goat anti-rabbit antibody at room temperature for 2 h. After PBST washing, the slices were sealed with glycerol and photographed under a fluorescence microscope.
2.11 Statistical Analysis
Statistical analyses were performed using GraphPad Prism software version 8.0 (GraphPad Software, La Jolla, CA, USA). All measurement data were reported as means ± standard deviations. One-way ANOVA was used to compare the mean difference of two or more groups. P-values of <0.05 were considered statistically significant.