2.1 Clinical samples
Nineteen patients with high-grade gliomas were selected from the Department of Neurosurgery of Ningxia Medical University General Hospital (March 2015 to November 2018, all confirmed by postoperative pathology). The patients included, 9 males and 10 females, aged 7-68 years, with an average age of 45.6 years. Before multivoxel 1H-MRS examination, all patients did not receive radiotherapy and chemotherapy, and had no history of brain injury and/or brain surgery. This study has been reviewed by the research ethics committee of Ningxia Medical University, and informed consent forms were signed with patients or their families before operation.
2.2 Image processing
Using signa HDX 3.0T superconducting MRI system made by GE company in the United States, multi voxel 1H-MRS was performed after conventional MRI plain scans and enhanced scans. MRI was used to determine the location of the lesion. The multi voxel 1H-MRS image and structure image data were transmitted to the post-processing workstation through LAN. The multi voxel analysis is carried out for each layer by using the self-contained software, and a 3D chemical displacement imaging is generated and converted into gray value image. The image contains standardized Cho / Cr information, and the Cho / Cr level of each voxel is represented by pseudo color image. Select the highest and lowest points of Cho / Cr as the region of interest and save them in DICOM format. The saved MRS images were imported into 3D interface, reconstructed and saved in axial direction. After being imported into navigation workstation, MRS sequences and structural images were fused and reconstructed, and the reconstructed sequences were exported to cranial software. In the fusion interface, the conventional anatomical sequences and MRS sequences were combined and multivoxel 1 H-MRS was added. The images were accurately fused into the intraoperative neuro-navigation system. With the aid of neuro=navigation, the highest and lowest Cho / Cr levels were accurately selected, and the tumor tissues in these two regions were taken as tissue samples, which were Cho / Cr high metabolism group and Cho / Cr low metabolism group, respectively.
2.3 Quantitative real time PCR
Total RNA was extracted from tumor tissue samples of Cho / Cr high metabolism group and Cho / Cr low metabolism group by Omega Kit (Omega , USA), and SYBR PremixExTaqTM. Gamma kit (Takara , Japan)was used to reverse the total RNA into cDNA, and then real-time PCRwas performed. The target gene CHKA primer sequence: upstream 5 '- CGGAAAAGTGCTCCTGCGGCT-3', downstream 5 '- AACCAAAGCTGTGCAGCCAA-3'. β - actin was selected as the internal reference gene. Samples were added according to the 20 μ l PCR system in the instruction manual and tested on the computer.
2.4 Western-blot Testing
The protein from Cho/Cr high metabolic group and Cho/Cr low metabolic group was extracted by full protein extraction kit and quantified by BCA. SDS-PAGE gel electrophoresis was carried out on the 40 micron g/ pore size. After electrophoresis, the protein on the gel was transferred to PVDF membrane at 300mA and 45 minutes. PVDF membrane was placed in 5% skimmed milk powder and sealed at room temperature for 2 hours then, it was added into CHKA primary antibody (Abcam , USA) for overnight on a 4 ℃ shaking table. The next day, the PVDF membrane was washed 5 times with TBST for 5 minutes each time. The second antibody of the corresponding species was incubated for 2 hours. After cleaning 5 times with TBST, the ECL emitted light.
2.5 Cell culture and lentiviral transfection
U251 cells (Shanghai cell bank of Chinese Academy of Sciences) were taken out from liquid nitrogen and placed in a 37℃ constant temperature water bath for 3-5min for resuscitation. The cell suspension in the cryopreserved tube was inhaled into a centrifuge tube containing 3ml culture medium (GIBCO ,USA) in an ultra-clean bench. Centrifugation was performed at 1000 RMP for 5 min. the supernatant was discarded. The cells at the bottom of the tube were used with 10% fetal bovine serum (GIBCO, USA) The DMEM with 1% double antibody was resuspended and seeded into a culture bottle, and then cultured in a cell incubator containing 5% CO2 at 37℃. U251 cells in logarithmic growth phase were seeded in a 6-well plate at the density of 1×105. After 24 hours, CHKA silencing lentivirus diluted with DMEM and its control lentivirus were added. After 12 hours of incubation, the DMEM medium containing 10% fetal bovine serum was replaced. After 48 hours of culture, the fluorescence of transfected cells was observed by fluorescence microscope. The cells were screened with puromycin (2 μ g / ml), and then identified by real-time PCR and Western blot.
2.6 Clonal activity was detected by clonal assay
The cells in logarithmic growth phase of CHKA group and vector group were digested and inoculated into a 6-well plate containing 2 ml medium at a density of 200 cells / well. The cells were evenly inoculated by shaking gently and then cultured in a cell incubator with 5% CO2 at 37℃ for 2-3 weeks. The cells were observed every 3 days. When cell clones were visible in the 6-well plate, the culture was terminated and the supernatant was discarded The bacteria were washed twice with PBS, fixed for 15 min with 4% paraformaldehyde, stained with 0.1% crystal violet solution for 30 min, and stained with 0.2% crystal violet solution for 30 min. finally, the cell clone formation rate was calculated.
2.7 CCK8 was used to detect cell proliferation
The cells were digested into single cells by trypsin and diluted to cell suspension with density of 3×104 cells / ml in culture medium. 100μ L cell suspension was inoculated into 96 well plate and placed in 37℃ constant temperature cell incubator containing 5% CO2 for 4H to make the cells adhere to the wall. DMSO were added to each treatment hole of 96 hole plate and MN58b with different dosages of DMSO at 10μm, 20μm and 40μm, were cultured in the cell incubator for 24 h, 48 h, 72 h and 96 h. Then 10μ CCK8 solution was added into each treatment hole of 96 well plate, and then cultured in the incubator for 2 h. The absorbance values of each well at 450 nm at different time points were measured by enzyme labeled instrument.
2.8 Transwell invasion assay was used to detect the invasion ability of the cells
The diluted Matrigel matrix glue was spread on the upper chamber of the small chamber, and the treated cells were diluted to 2×105 cells / ml with serum-free DMEM medium. 200 μL cell suspension was added into the upper chamber and the lower chamber was added with DMEM medium containing 20% fetal bovine serum. The cells in the upper chamber were wiped off with cotton swab and fixed with 4% polymethanol for 30 min, and stained with 0.1% crystal violet solution for 30 min. The cells were then observed under a microscope and photographed.
2.9 Animal model and tumor cell implantation
Six-week-old female nude mice (Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., 18-22g) were raised in a SPF environment for one week. U251 cells suspended in serum-free medium (1x107) were injected into the axilla of the right forelimb of each mouse. When the volume of the tumor on the right side of the mouse reached about 100mm3, the mice were divided into two groups, 6 in each group. The experimental group was intraperitoneally injected with MN58b (2mg/kg/ days), and the control group was injected with normal saline. After 14 days of continuous treatment, all nude mice were killed and samples were taken as required.
The paraffin-embedded tumor specimens were cut into 4μm thick slices, which were routinely dewaxed and placed in sodium citrate buffer for antigen repair, cooled naturally for 2 hours, incubated with 3% hydrogen peroxide solution at room temperature for 10 minutes, and sealed with goat serum. Removing the sealing solution, the primary antibody E-cadherin (Servicebio,1:300), N-cadherin (Servicebio,1:300) and Vimentin (Servicebio,1:200) were incubated overnight at 4 ℃, and the second antibody (Servicebio,1:500) was added the next day. After DAB staining, the expression of E-cadherin, N-cadherin and Vimentin in glioma was observed under microscope.
SPSS 24.0 software was used for statistical analysis. The data results were expressed as mean ± standard deviation ( ± s). t-Test was used for comparison of two samples, one-way ANOVA was used for diversity comparison, and SNK-q test was used for comparison between two samples. The difference was statistically significant (P<0.05).