Patients and tissue samples
Tumor tissue microarray was afforded by the Affiliated Hospital of Xuzhou Medical University. All patients involved in this study provided written informed consent. According to the WHO, the tumors were divided according to the pathological grade as follows: 237 cases with grades I-II and 195 cases with grades III-IV.
Immunohistochemistry (IHC) and histopathological assessments
IHC was uniformly performed to evaluate the SMYD2 protein expression in the glioma samples. Samples using the Bond Polymer Refine Detection System (Leica Biosystems, Wetzlar, Germany). The immune-reactive score (IRS), which represents the intensity and quantity the stained cells, was implemented to quantify the staining level of SMYD2. The SMYD2 staining intensity was scored as follows: 1, negative; 2, weak; 3, moderate; and 4, strong. The proportion of the positively stained cells was defined as follows: 1, 0%-25%; 2, 26%-50%; 3, 51%-75%; 4, 76%-100%. The immunoreactive score (IRS) was calculated by multiplying the score of the staining intensity and the proportion of positive cells. Samples with an IRS score of <=4 was considered as those with low SMYD2 expression and samples with an IRS score of >5 was considered as those with high SMYD2 expression.
Cell lines and cell culture
U251 and U87 human glioma cell lines were purchased from the Cell Bank of the Type Culture Collection Committee of the Chinese Academy of Sciences. These glioma cells were cultured in DMEM medium supplemented with 10% fetal bovine serum and were maintained at 37℃ in a humidified incubator with 5% CO2.
Antibodies, reagents and plasmid
Antibodies against the following proteins were used: γ-H2AX (ab81299) from Abcam (Cambridge, UK); Histone H3 (4499) and Di-Methyl-Histone H3 (Lys 36) (2901) from Cell Signaling Technology (Danvers, MA, USA); SMYD2 (21290-1-AP) from Proteintech (Rosemont, IL, USA); Methyl P53 (Lys 370) from Immunoway Biotechnology (Plano, TX, USA); GAPDH (AG019), Bax (AF1270), Bcl2 (AF6285), CyclinD1 (AF1183), P53 (AF1162), COL1A1 (AF6524), N-Cadherin (AF5237) and E-Cadherin (AF6759) from Beyotime Biotechnology (Shanghai, China). The following reagents were used in this research: AZ505 (HY-15226), Cisplatin (HY-17394) and Temozolomide (HY-17364) from MedChemExpress (Monmouth Junction, NJ, USA). Plasmid of SMYD2 (HG11093-NF) was purchased from SinoBiological (Beijing, China).
Quantitative reverse transcription PCR (RT-qPCR)
To evaluate the differentially expressed mRNAs in AZ505-treated cells relative to control cells, total cellular RNA was extracted using TRIzon reagent (CW0580, Cwbio, Beijing, China) and cDNAs were synthesized using PrimeScript™ RT Master Mix (RR036A, Takara). Realtime PCR was performed using SYBR Green Realtime PCR Master Mix (RR430, Takara) and Cobas z 480 (Roche, Basel, Switzerland). The primers for SMYD2, BAX, GADD45, P21, COL1A1, MMP7 and β-actin were synthesized by TSINGKE Biological Technology (Beijing, China). β-actin was served as an internal reference of RNA integrity. The sequences are as follows:
SMYD2-F 5’-CTCCAAGCATCTCGGATTCCC-3’
SMYD2-R 5’-TGCAACATCAGGAAATATCGCTG-3’
BAX-F 5’-TTTGCTTCAGGGTTTCATCC-3’
BAX-R 5’-CAGTTGAAGTTGCCGTCAGA-3’
GADD45-F 5’-GGATGCCCTGGAGGAAGTGCT-3’
GADD45-R 5’-GGCAGGATCCTTCCATTGAGATGAATGTG-3’
P21-F 5’-TGTACCCTTGTGCCTCGCTC-3’
P21-R 5’-TGGAGAAGATCAGCCGGCGT-3’
COL1A1-F 5’-GAAGACATCCCACCAATCACC-3’
COL1A1-R 5’-TCGTCACAGATCACGTCATCG-3’
MMP7-F 5’-ACAGGCTCAGGACTATCTCAAG-3’
MMP7-R 5’-ACATTCCAGTTATAGGTAGGCC-3’
β-actin-F 5’-ACTCTTCCAGCCTTCCTTCC-3’
β-actin-R 5’-CGTCATACTCCTGCTTGCTG-3’
Western blot
The cells were harvested and rinsed twice with PBS. All protein samples were denatured and separated on 8-12% SDS-polyacrylamide gels and transferred to polyvinylidene fluoride membranes. Membranes were blocked with 5% milk in T-BST at room temperature for 1 h and incubated with primary antibody overnight at 4 ℃. After washing 3 times with T-BST, membranes were incubated with secondary antibodies for 1 h at room temperature. Proteins visualized using the Electrochemiluminescence (ECL) and detected in The ChemiDoc MP system (Bio-Rad, Hercules, USA).
Migration assays
This assay was conducted using a transwell chamber (3422, Costar) with a pore size of 8 μm. The upper chamber was filled with 5x104 cells in a serum-free medium. After incubation for 24 h for migration, the cells in the upper chamber were carefully removed with a cotton swab, the cells that traversed in the membrane were fixed in methanol and stained with crystal violet, and the permeating cells were calculated and photographed under light microscopy.
Cell proliferation assay
Cells were plated onto 96-well plates at a density of 2000 cells per well. After 24 h, cells were treated with DMSO or AZ505 and then cultured for 24, 48, 72 and 96 h, respectively. At the same time point on these 4 days, CCK-8 solution (C0042, Beyotime Biotechnology, China) was added to the wells needed to be measured. After incubation for 1 h at 37 ˚C, the proliferation rate was evaluated based on the increase in the absorbances at 450 nm.
Wound healing assay
Cells were plated onto 6-well plates at 100% density and cultured for 24 h. Then a rectangular lesion was created using a plastic pipette tip, and the monolayer was irrigated twice with PBS and incubated in serum-free media with AZ505 or DMSO. At the designated time, nine randomly selected fields at the lesion border were determined for shooting under an inverted microscope.
Cell apoptosis assay
Cells were plated onto 6-well plates at a density of 70-80% for 24 h. For analyzing the apoptosis induced by AZ505, cells were treated with DMSO or AZ505. For analyzing drug sensitivity induced by AZ505, cells were treated with DMSO, cisplatin or the combination of AZ505 and cisplatin. For analyzing drug resistance induced by the knockdown of STUB1, cells were treated with si-NC or si-STUB1 for 24 h, then cells were treated with DMSO or cisplatin. After 24 h, cells were collected and were marked using 300 μL Annexin V-FITC binding reagent containing 6 μL propidium iodide (PI) and 3 μL Annexin V-FITC (C1062L, Beyotime Biotechnology, China) for 15 min at room temperature. The apoptosis rate was measured using a Flow cytometer (EasyCell 204A1/206A1, Wellgrow, China).
Active oxygen detection assay
Cells were plated onto 6-well plates at a density of 70-80% for 24 h. For analyzing the apoptosis induced by AZ505, cells were treated with DMSO or AZ505. For analyzing drug sensitivity induced by AZ505, cells were treated with DMSO, cisplatin or the combination of AZ505 and cisplatin. After 24 h, cells were collected and washed for 3 times using PBS. 500 μL DMEM containing 10 μM DCFH-DA was added to cells. The mixture was incubated at 37 ˚C for 20 min and then washed using PBS for 3 times to fully remove the DCFH-DA that has not entered the cells. Cells were resuspended using 300 μL PBS. And the active oxygen level was detected using a Flow cytometer (EasyCell 204A1/206A1, Wellgrow, China).
Bioinformatics analyses
AZ505-treated and control U251 cells were collected for RNA sequencing conducted by CapitalBio corporation (Beijing, China). Limma package in the R software was used for the differential mRNA expression analysis. Significantly dysregulated mRNAs were defined using a cutoff |log2fold-change| value of >1 and P value of <0.05. The identified differential expressed genes (DEGs) were subjected to both Gene Ontology (GO) and Kyoto Encyclopedia of Gene (KEGG) pathway analyses using ClusterProfiler package. And the Gene Set Enrichment Analysis (GSEA) were analyzed using all detected genes on the GSEA software (version 4.1.0). The protein-protein interaction (PPI) analysis was produced in STRING website (https://string-db.org/), and the results were then imported into Cytoscape software (https://www.cytoscape.org). ClueGo app was used to GO and KEGG pathway analyses. Then, hub genes were predicted using degree algorithm of cytoHubba app. The prognostic values of these hub genes were assessed in GEPIA2 website (http://gepia2.cancer-pku.cn/#index).
The prognostic analysis of SMYD2, COL1A1 and STUB1 were assessed in GEPIA2 and CGGA website (http://cgga.org.cn/index.jsp). The expression level in different stage of glioma of SMYD2, COL1A1 and STUB1 were analyzed in CGGA website.
Statistical analysis
Statistical analyses were performed using the GraphPad Prism 9 for Mac (GraphPad Prism Software Inc., San Diego, CA, USA). Comparisons between two groups were performed using an unpaired Student’s t test. One-way analysis of variance (ANOVA) followed by post-hoc test was used for multiple comparisons. These data are presented as the means + standard deviations (SDs), and P < 0.05 was considered to reflect a statistically significant difference.