Patients and samples
Glioma tissues and normal brain tissues were collected from the Department of Neurosurgery in Renmin Hospital of Wuhan University from July 2015 to July 2018. Normal brain tissues were obtained during the operation of severe traumatic brain injury with the informed consent of the patients requiring surgery. A total of 92 paraffin-embedded glioma tissues were used for immunohistochemical staining. For Western blot, 8 frozen glioma tissues and 5 frozen normal brain tissues stored at − 80 °C was evaluated. None of the patients received any chemotherapy or radiation therapy before surgery. All patients signed an informed consent form, and the study was subject to approval by the Ethics Committee of Renmin Hospital of Wuhan University (approval number: 2012LKSZ (010) H).
TMCO1 (ARP49429_P050) antibody was purchased from Aviva Systems Biology. E-cadherin(20874-1-AP), N-cadherin(22018-1-AP), β-actin（66009-1-Ig），Vimentin (10366-1-AP), Snai1(13099-1-AP),caspase3(19677-1-AP),MMP2(10373-2-AP), and Bcl2 (12789-1-AP) antibodies were purchased from Proteintech.
In order to evaluated the expression and prognostic role of TMCO1 in gliomas, we download normalized RSEM gene-level RNAseq and corresponding clinical data of TCGA, CGGA, Gill and Rembrandt datasets from Gliovis database (http://gliovis.bioinfo.cnio.es/). Gliovis website is an important data visualization and analysis platform for studying brain tumors.
Immunohistochemical (IHC) staining
A total of 92 glioma tissues and 10 normal brain tissues were fixed with 4% paraformaldehyde and embedded in paraffin. The slices were hydrated in xylene and ethanol with different concentrations (100%, 95%, 75%). Afterwards, 3% H2O2 was added and incubated for 10 minutes after washing three times with PBS buffer. Then, the sections were subjected to antigen retrieval in 10 mM sodium citrate (pH, 6.0) at 95℃ for 10 mins and cooled naturally. Subsequently, the sections were blocked with 1% bovine serum albumin (BSA) for 30 minutes, and incubated with primary antibody at 4 ℃ overnight. The next day, the sections were incubated with HRP-labelled secondary antibody (Antgene, China) for 1hour and stained with DAB kit and hematoxylin. Finally, images were obtained using an Olympus BX51 microscope (Olympus, Japan). The intensity of IHC was divided into: 0, 1, 2, 3 points, which indicated background staining, faint staining, moderate staining and strong staining respectively. Two independent pathologists examined and scored. If they have different opinions, a third pathologist will be added for scoring. IHC score of 0-1 was defined as low expression group, and score of 2-3 was divided into high expression group.
Cell culture and transfection
U251 and U87 cell lines were purchased from the cell bank of the Chinese Academy of Sciences (Shanghai, China). All cell lines were cultured at 37 ° C in a humid atmosphere of 5% CO2 using Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Gibco, USA). TMCO1-shRNA was purchased from Genechem (Shanghai, China). Cells were transfected with 2.0 ug plasmid per well in 6-well plates using transfected with lipo3000 (Invitrogen, USA) according to the manufacturer’s instructions. Fresh DMEM supplemented with 10% FBS was added to 6-well plates after 24 hours of transfection, and the cells were harvested for subsequent experiments after 48 hours of transfection.
U87 and U251 were lysed on ice with modified RIPA buffer (Beyotime, China) for about 30 minutes, and then centrifuged at 12,000 rpm at 4℃ for 15 minutes. For cryopreserved glioma tissues, we added 1.5ml of modified RIPA buffer per 100 mg of tissue. The sample concentration was quantitatively determined by BCA protein assay. The lysate was mixed with loading buffer (Beyotime, China) and heated at 100°C for 5 minutes. An equal amount of protein was loaded into 10 or 12% SDS-PAGE, and then transferred to a PVDF membrane (Millipore, Germany). Next, the PVDF membrane was blocked in 5% skimmed milk for 1 hour, and incubated with the primary antibody at 4°C overnight. Then, the membrane was incubated with a secondary antibody (Antgene, Chian,1:3000) at room temperature for 1 hour, and then visualized using ChemiDoc™ Touch Imaging System (BIO RAD,China)
Wound healing and transwell assay
Cells were seeded in a 6-well plate and cultured for a certain time to reach 80% confluence. A sterile pipette tip was used to scratch a linear wound and washed away floating cells with PBS buffer, and then serum free DMEM was added for further culturing. Wound healing images were captured at certain time using an inverted microscope (Olympus BX51, Japan) and ImageJ software was used in the subsequent analysis. For transwell assay, U87 and U251 cells were seeded into the upper chambers (Corning, USA) precoated with Matrigel (R&D, USA) after transfection for 48 hours, then 200 ul serum-free DMEM medium was added. The lower chamber was filled with 600 μl of DMEM containing 10% FBS. Transwell chambers were placed in an incubator (37 °C,5% CO2) for 24 h. Cells were fixed with 4% paraformaldehyde for 30mins, stained with 0.2% crystal violet for 15mins and counted under an inverted microscope (Olympus BX51, Japan).
Clone formation and cell counting kit-8 (CCK8) assay
After transfection for 48hours, 500 glioma cells per well were seeded in 6-well plates and cultured in complete medium until there was obvious single-cell colony formation. Subsequently, cells were fixed with 4% paraformaldehyde for 15mins and stained with 0.2 crystal violet for 15mins. For CCK8 assay,4000 cells were resuspended in 100 μl complete medium and then seeded to a 96 well plate. The proliferation capacity of the cells was assessed at specific times according to the instructions of the CCK8 kit (Dojindo, Japan)
Flow cytometric analysis
Annexin V-PE/7- ADD kit (Becton Dickinson, USA) were used to measure the apoptosis of glioma cells. According to the manufacturer’s instruction, cells were harvested after transfection for 48 hours and washed three times with PBS buffer. The apoptosis of samples was measured by FACS Calibur flow cytometer (BD Biosciences, USA) after cells were stained with PE and 7- ADD for 15 mins under dark conditions.
Statistical analysis was performed using GraphPad Prism 8.0.2. software. All datas were presented as mean ± standard deviation (SD). Student’s t-test was used to analyze the differences between two groups. One-way analysis of variance (ANOVA) was used for the comparison among three or more groups, and Tukey’s multiple comparisons test was performed to test differences between groups if analysis of variance was significant. Patients were divided into high and low groups according to the 50% cutoff point of TMCO1 expression and Kaplan–Meier survival analysis was used to analyzed significance between groups. The p value less than 0.05 was considered significant.