2.1 Differential gene expression analysis
Expression profile of GSE68029(28) and GSE16011(29) were extracted from Gene expression omnibus (GEO). These transcriptome profiles were preprocessed as previously described(30). Gene expression datasets of TCGA GBM was extracted from GDC Data Portal (https://portal.gdc.cancer.gov/). The limma package(31) was used for identifying the differentially expressed genes in these datasets. The expression difference of individual gene was defined by log2(Fold change) and adjusted P value, in which log2FC<-1 with an adjusted P value < 0.05 was defined as a down-regulated gene. Venn diagram was carried out to illustrate the overlapping down-regulated genes in these datasets.
2.2 Ethical statement and patient samples
This study was approved by the Scientific Ethics Committee at the First Affiliated Hospital of Xi’an Jiaotong University (approval no. 2016-18). All the patients’ samples used in this study have obtained necessary consent, and all samples were embedded in paraffin blocks as previously described(30).
2.3 Immunohistochemistry (IHC) and global immunohistochemistry score (GIS)
Immunohistochemistry was performed as previously described(32, 33). Anti-PLK2 primary antibodies were purchased from Abcam (cat. no. ab71311). Secondary antibodies including Goat anti-rabbit IgG (cat. no. ab97051, Abcam) and goat anti-mouse IgG (cat. no. ab205719, Abcam) were used in this study. Tissues embedded with paraffin were cut into 4-mm sections followed by deparaffinized, rehydrated, and stained with primary antibodies at 4℃ overnight. The slides were then incubated with secondary antibodies and stained with DAB. Consequently, the slides were counterstained with hematoxylin and images were taken under the light microscope.
The global immunohistochemistry score (GIS) was used to measure the relative expression of PLK2 in different samples. GIS was calculated by the following equation: immunoreactivity score = staining intensity score × positive cell score. The staining intensity score was scored as: 0, negative; 1, weakly positive; 2, moderately positive; and 3, strongly positive. The positive cell score was scored as: 0, negative; 1, <10% positive; 2, 11%‐50% positive; 3, 51%‐80% positive; and 4, >80% positive. Consequently, an immunoreactivity score >5 was considered as high expression, and ≤5 was defined as low expression.
2.4 Lentivirus production and transduction
Lentivirus production and transduction were performed as described previously(30). The lentiviral overexpression/knockdown PLK2 was designed and synthesized by Genechem (Shanghai, China). These lentiviruses were introduced into U87 and U251 cell lines according to manufacturer’s instruction. Stable clones transduced with PLK2 and shPLK2 were selected for 4 weeks by puromycin. Lentiviral particles packaging the shRNA are targeting PLK2 (5′-TAGTCAAGTGACGGTGCTG-3′) and the scramble control (5′-TTCTCCGAACGTGTCACGT-3′).
2.5 Cell culture, in vitro cell proliferation and cell viability assay
GBM cell lines including U87 and U251 were purchased from Cell bank, Type culture collection, Chinese Academy of Sciences(Xi’an China) as previously described(30). Cells were cultured in DMEM-F12 containing 10% FBS and antibiotics (1% penicillin and streptomycin). U87 and U251 cells were cultured in a humidified condition containing 5% CO2 at 37℃.
For in vitro cell proliferation assay, cells were suspended adequately before seeded into 96-well plates at a density of 1 × 103 cells/well. Cell number was counted by using alamarBlue following the manufacturer’s instruction after indicated treatments.
Cell viability assays were conducted as previously described(30). In brief, cell number was calculated by cell counter with trypan blue, cells were then seeded into 96-cell plates after suspended adequately at a density of 2 x 103 cells/100 uL per well. After 24 hours of culturing, U87 and U251 cell lines received indicated in vitro chemotherapy using corresponding concentration of TMZ. At last, cell number was counted by alamarBlue and IC50 was calculated by SPSS 22.0.
2.6 Quantitative RT-PCR (qRT-PCR) and Western blot analysis
The Quantitative RT-PCR assays were conducted as previously described(30). In brief, total RNA was extracted using RNeasy mini kits according to the manufacturer’s instruction, and the concentration of RNA was determined by Nanodrop 2000. qRT-PCR was performed following the synthesis of cDNA according to the standard protocols. GAPDH was used as an internal control. Relative mRNA expressions were calculated by 2‐ΔΔt method. The primer sequences were listed as below:
PLK2: forward, 5′-GCTGATGTCTGGCTGTTCATCAG-3′ and reverse, 5′-CTTCCCTGTAGATCTCACA GTG-3′;
HES1: forward, 5′-AGTGAAGCACCTCCGGAAC-3′ and reverse, 5′-TCACCTCGTTCATGCACTC-3′
c-MYC: forward, 5’- AAACACAAACTTGAACAGCTAC-3’ and reverse, 5’- ATTTGAGGCAGTTTACATTATGG-3’
p21: forward, 5’- GGCATAGAAGAGGCTGGTGG-3’ and reverse, 5’- ATGGCGCCTGAACAGAAGAA-3’
Cyclin D3: forward, 5’- AAACTTGGCTGAGCAGAGCA-3’ and reverse, 5’- GAACAGAGCCAGTCTCCACC-3’
GAPDH: forward, 5′-ACCCAGAAGACTGTGGATGG-3′ and reverse, 5′-TTCAGC TCAGGGATGACCTT-3′.
Western blot analyses were performed as previously described(32). Antibodies used in this study were shown as below: Anti-PLK2 primary antibody was purchased from Abcam (cat. no. ab71311). Anti-β-actin antibody was purchased from Abcam (cat. no. ab115777). Anti-NOTCH1, anti-NOTCH2, anti-HES1, anti-c-MYC and anti-cyclin D3 primary antibodies were bought from Cell Signaling Technology (CST) with the catalog number of #4147, #5732, #11988, #18583 and #2936, respectively. Additionally, Anti‐Rabbit IgG and Anti‐Mouse‐IgG were purchased from CST (cat. no. #7074 and #7076, respectively).
2.7 Colony formation assay and wound healing assay
Colony formation assays were conducted to detect the tumorigenic potential of U87 and U251 cell lines. Different numbers of stable U87 and U251 cells transduced with lentiviral PLK2 or vector were seeded into 6-well plates under indicating conditions. After cultured for 14 days to form colonies, these cells were fixed with methanol and stained with methylene blue.
The wound healing assay is used to study the migratory ability of U87 and U251 GBM cell lines. Cells were seeded in 6-well plates after adequately suspended. Afterwards, a sterile pipette tip was used to produce a wound line when cells reached the appropriate confluence. These cells were then washed at least 3 times to be cultured in FBS-free medium. Images were taken under inverted microscope at the same location. The leading edges were marked by white lines, and the relative distance of the borders was measure by Image J software.
2.8 The construction of TMZ resistant glioma cell lines
To construct TMZ resistant cell lines, U87 and U251 were gradually treated with increasing concentration of TMZ. In brief, the beginning concentration of TMZ was set according to the IC50 of U87 and U251 naïve cells. The medium was changed every two days to keep the concentration of TMZ at a stable level for two weeks. Afterwards, new IC50 was calculated using the pre-treated cell lines and the process was continued until the IC50 of TMZ reached the amount of 500uM. These cells were then used for subsequent experiments.
2.9 In vivo intracranial xenograft tumor models
The usage of experimental animals in this study was approved by the Ethics Committee of the School of Medicine, Xi’an Jiaotong University (approval no. 2016-085). In vivo xenograft model was constructed by using 6-week-old female nude mice. U87 and U251 cells transduced with indicating lentiviruses was suspended and diluted to the density of 1 x 105 cells in 2 uL PBS then slowly injected into the nude mice brains. Each group of treatment contains five mice, and they were monitored until the following symptoms were observed: unsteady gait, arched back, more than 10% weight loss or leg paralysis.
2.10 Flow cytometry
Flow cytometry assays were performed as previously described(30). U87 TMZ resistant cells were transfected with either empty vector or plk2 overexpression lentivirus then treated with 200uM TMZ for 24 h. DMSO were used as a negative control. The Alexa Fluor® 488 Annexin V/Dead Cell Apoptosis kit was used strictly following the manufacturer’s protocol to measure the cell apoptotic rate.
2.11 Gene set enrichment analysis (GSEA)
The transcriptome profiles of previously mentioned datasets were read into R programming and preprocessed by background correction, gene ID transformation and normalization. These data were then ordered by the expression level of PLK2 to divide all samples into two groups (PLK2high and PLK2low) by quartile cutoff. Afterwards, these profiles were uploaded into the GSEA software(34) strictly following the guideline of the software to elucidate the enriched KEGG pathways that are significantly enriched in PLK2low groups with the number of permutations set at 1000.
2.12 Glutathione S-transferase (GST) pull-down assay
The ubiquitination level of Notch1 was determined by GST-mediated pull-down assays (Thermo Scientific, Rockford, IL). Briefly, GST-tagged Notch1 was transfected into indicated cell lines (isogenic PLK2 overexpressed cells or their negative control) by lipofectamine according to manufacturer’s instruction. After 24 hours, cell lysates were collected and incubated with GST beads for 1h. Then, GST complexes were washed extensively with lysate buffer and used to perform western blot assays.
2.13 Statistical analysis
All the results in this study are exhibited as mean ± SD (Standard deviation) as described previously(35). The number of independent replications is presented in the figure legends. Two-tailed t tests were conducted to evaluate statistical differences and One-way ANOVA following Dunnett’s post-test was used to compare the statistics in more than two groups. Kaplan-Meier survival analyses were performed using log-rank test. In addition, all statistical analysis was performed by GraphPad Prism 7 or SPSS 22.0 software, and statistical significance was defined as a two-sided P value less than .05 unless specifically indicated.