The glioma samples were taken from patients with glioma in the Department of Neurosurgery, the Affiliated Hospital of Guizhou Medical University. A total of 81 tissues were involved in this study, including 10 normal brain tissues. The experiment involving specimens in this study obtained the consent of the patient and passed the approval of the ethics committee of this hospital.
Human glioma cell lines (U118, A172, LN229, U251) were purchased from the Shanghai Chinese Academy of Sciences Cell Bank. All cells were cultured in DMEM high-sugar medium and FBS (10%), and then placed in a 37°C, 5% CO2 cell incubator.
Real-time fluorescence quantitative PCR
Fully lyse cells or tissues with Trizol reagent, RNA extraction kit (AM1931) was used to extract cells and tissues separately according to the instructions. Follow the steps of the RNA Reverse Transcription Kit (Applied Biosystems™,4368813) to gradually complete the reverse transcription of RNA. The Q-PCR reaction was performed according to the requirements of SYBR Green Master Mix Kit (Applied Biosystems™,4432346), results were calculated according to the Ct value (2ΔΔCt). The primer involved were as follows: β-actin (internal control) primer (forward primer, 5’-CATGTACGTTGCTATCCAGGC-3’; reverse primer, 5’-CTCCTTAATGTCACGCACGAT-3’); TPPP3 primer (forward primer, 5’-GGTCCATTCCTGCGTCGTTC-3’; reverse primer, 5’-GCCCAGTTCTTGCCATTCATC-3. All reagents and kits were purchased from Thermo Scientific.
Western blot analysis
An appropriate amount of RIPA lysate was used to lysate cells or tissues and extract proteins. Protein concentration was measured with BCA kit (Abcam, ab102536). Protein samples and protein markers were loaded in 10% separation gel and then transferred to 0.45-µm PVDF membranes (Millipore, 42029053) activated with methanol. Then membrane was blocked in 5% non-fat milk for 2h and incubated with primary antibody (Abcam, USA) at 4°C overnight. Then placed into the corresponding secondary antibody (Abcam, USA,1:5000) box and incubated with a shaker at room temperature for 2h. Finally, the ECL working solution (Thermo Scientific™, USA) was prepared according to 1:1, and the membrane was exposed in the chemiluminescence instrument, the results were analyzed by Image J. The involved primary antibodies were as follow and concentration was diluted according to product instructions: TPPP3, N-cadherin, E-cadherin ,Vimentin, Snail 1,Slug,Twist 1,ZEB 1,β-catenin(all from Abcam).
The cells were fixed with 4% paraformaldehyde in the petri dish for 15min. Then add 0.2%Triton X-100(Sigma-Aldrich, Germany) 200uL/ dish permeability 10min. Wash PBS 3 times /2min, add 200uL sealing solution/dish at room temperature for 1h. Dilute the primary antibody (Abcam, USA) with PBS in accordance with the required proportion and incubate at 4℃ overnight. The fluorescence secondary antibody (Abcam, USA) was diluted well, and 200uL/ dish was added, wrapped in tin foil to avoid light, and sealed at room temperature for 2h. 200uL/ dish of DAPI (Abcam,100ng/mL) was added and dyed at room temperature for 10min, with the whole process shielded from light. Finally, photographs are taken under a confocal laser microscope.
The cells were counted and added with DMEM high glucose medium containing 10% FBS to adjust the cell concentration to 200,000 cells/well. The cells were cultured in cell incubator. When the cells grew to 80%~90%, mark "+" at the bottom of the hole with 10uL spear head, remove the old culture medium, then add 900uL DMEM culture medium to each well, take photos with fluorescence microscope. The cells were photographed again at the same location 24 hours later, and their mobility was compared. Cell migration rate = experimental group migration area/control group migration area.
Add 50µL Matrigel (Corning, USA) to the upper chamber of each Transwell cell (diluted 1:8 in DMEM serum-free medium) and place in an incubator for 30 min. The cell concentration was adjusted to 100,000 cells/well. Transewell added 600uL DMEM containing 1% FBS into the 24-well plate in the lower chamber, and 200uL DMEM medium containing 100,000 cells into the upper chamber (Corning, CLS3470), and cultured in the cell incubator for 24h. The cells were then placed in a 24-well plate containing 600uL 4% paraformaldehyde for 30min and stained with 1% crystal violet (Sigma-Aldrich, V5265) for 30min. Gently wipe the upper chamber with a cotton swab, dry it, and take photos with a fluorescent microscope for counting.
Cell count kit-8(CCK8) assay
When the cells grew to 70%-80% and fused, the adherent cells were digested with trypsin. the adherent cells were collected and counted to prepare cell suspension with a density of 5×104/ mL, and 100µL cell suspension was prepared in a 96-well plate. The culture plate was pre-cultured in the incubator for 24 hours. According to the instruction of CCK8 kit (Dojindo, Japan), 10ul working solution was added to each well. After incubation for one hour, the absorbance at 450nm was measured with a microplate reader.
The prepared paraffin sections were stained by Immunohistochemistry. The staining was completed according to the description of the immunohistochemical staining kit (CST, SignalStain® Boost) instructions. The brief steps are as follows: The dewaxed tissue sections were placed in anhydrous ethanol for 15 minutes, 95%, 90%, 80% and 75% ethanol for 10 minutes successively, and finally placed in ddH2O for 15 minutes for dehydration. The sections were immersed in 1 X citrate repair solution, then heated in the microwave oven until boiling, and kept at temperature (95°-98°) for 10 minutes. Then the sections were cooled for 30 minutes for antigen repair. After inactivation of endogenous peroxidase in tissues, the slices were closed and incubated in diluted primary and secondary antibodies. DAB chromogenic solution was added on the surface of the slices, and the reaction lasted until brownish yellow color appeared. The chromogenic solution was removed, and the slices were cleaned with PBS, then hematoxylin was added for redyeing for 3 minutes. Finally, it is observed and photographed under a microscope.
The stained sections were observed by two pathologists under a 200x microscope and evaluated independently. The immune response score was calculated by the following formula, IS = percentage (0, no target molecule expressed; 1, 1% ~ 10% was positive; 2,11% ~50% is positive; 3, > 50% is positive) x staining intensity (0, no color; 1, weak color rendering; 2, medium color display; 3, strong color). When the IS score is between 3 and 9, it means that the target protein is highly expressed, otherwise the target protein is considered to be low-expressed.
Small hairpin RNA (shRNA) lentiviruses
Cell lines were constructed using lentivirus transduction as described previously.Lentivirus (Genechem, China) was packaged in 293T cells and transfected with Lipofectamine 3000 liposome transfection reagent (Thermo Scientific, L3000001). The interference sequence of TPP3 as mentioned in the study of Li et al. sh1:5’-CCGGGCCAATGTGGGCGTCACTAAACTCGAGTTTAGTGACGCCCACATTGGCTTTTTG-3’, sh2:5’-CCGGCTGCTCGGGTCATCAACTATGCTCGAGCATAGTTGATGACCCGAGCAGTTTTTG-3’, sh3:5’-CCGGAGGAGAGCTTCCGCAAGTTTGCTCGAGCAAACTTGCGGAAGCTCTCCTTTTTTG-3.When the cells in the blank control die and the lentivirus-transfected cells are still alive, the selected cells are expanded and sub-cultured, and cells were collected to detect the expression of the target protein.
Xenograft mouse model
Animal experiments are approved by the Ethics committee and carried out in strict accordance with the requirements. Twenty-four 5-week-old female BALB/c-nude mice were selected from Shanghai experimental animal center of Chinese Academy of Sciences for xenotransplantation experiment. U251 cells (shCrtl, shTPPP3) with a cell density of 5x107 / mL were injected into the right flank of mice. The size of the tumor was measured with a vernier caliper every 5 days after tumor formation, the tumor volume = (width2 × length) / 2, the curve was plotted based on time - weight. After 30 days of subcutaneous inoculation, the nude mice were sacrificed by neck-method, and the tumor tissues in vivo were separated for weighing, photographing, and subsequent experiments.
In this study, GraphPad Prism software was used for statistical analysis of data. Student's test was used for significant differences among different groups. Kaplan-Meier survival curve and log-rank test were used to analyze survival differences. A P value less than 0.05 was considered statistically significant.