Public Data Analysis
The gene expression quantification data and clinical data of gastric carcinoma patients were downloaded from the GDC data portal (https://portal.gdc.cancer.gov/). The expression difference of CPXM1 in normal gastric tissue and gastric cancer tissue was verified in the GEPIA database (http://gepia.cancer-pku.cn). NetNGlyc 1.0 was used to predict putative N-Glycosylation sites (http://www.cbs.dtu.dk/services/NetNGlyc/)
GC patients and collection samples
From May 2019 to December 2019, 20 cases of fresh gastric cancer tissues and corresponding adjacent tissues were collected from the First Affiliated Hospital, Yijishan Hospital of Wannan Medical College. The tissues were collected and transported in dry ice, then stored in liquid nitrogen for a long time. The inclusion criteria of patients were as follows: 1, gastric cancer confirmed by gastroscopy and pathology; 2, patients without other tumors; 3, patients without chemotherapy or radiotherapy. Informed consent was signed by all patients participating in the study. The study was approved by the ethics committee of the First Affiliated Hospital, Yijishan Hospital of Wannan Medical College.
GC tissues HIC and bioinformatics analysis
The detailed operation of this part is as described.
Five gastric cancer cell lines ( SGC-7901, MGC-803, BGC-823, HGC-27, MKN-45) and GES-1 were purchased from Shanghai cell bank of the Chinese Academy of Sciences and cultured in our laboratory for long-term preservation. The cells were cultured in RPMI-1640 (Hyclone, USA) supplemented with 10% fetal bovine serum (FPS, Beyotime, China) at 37 ℃ in a humidified cell incubator containing 5% CO2.
The GC cells were cultured on the 6-well plates for 24 hours to make the density reach 80% confluence. Lipofectamine 3000 reagent (L3000015, Thermo Fisher, USA) was diluted in opti-MEM medium (31985062, Thermo Fisher, USA). The plasmid (GenePharma, China) was diluted with opti-MEM medium, and then P3000 reagent was added. The two reagents need to be well mixed. The diluted plasmids were added into each tube of diluted Lipofectamine 3000 reagent at a ratio of 1:1, and then incubated for 15 minutes. The mixed reagent was added into the cells which were transfected for 48 hours. Western bolting was used to detect protein expression to determine the transfection efficiency.
Lentivirus transfection and establishment of stable cell line
The GC cells were cultured on the 96-well plates for 24 hours to make the density reach 20% confluence. 10 µl lentivirus stock solution (GenePharma, China) was added to each well. After 48–72 hours infection, the infection efficiency was observed under fluorescence microscope. When the infection rate was 90%, the cells from 96 well plate were transferred to 24 well plate. At this time, the cells were cultured in 2 ug/ml purinomycin medium (P8230, Solarbio, China) for 2 weeks until the fluorescence expression rate of the cells was stable at 90%. Western bolting was used to detect protein expression to determine the transfection efficiency.
Cell invasion and migration assay
For cell migration assay, 1 × 105 cells were plated in the upper chamber of 24-well transwell plates (8-µm pore polycarbonate membrane, Coring, USA) with 200 µl serum-free medium, while the lower chamber loaded 600 µl RPMI-1640 medium containing 20% FBS. For cell invasion assay, the upper chamber of 24-well transwell plates were previously coated with MixGel ECM (E0282, Sigma, USA). After invasion and migration for 24 h, the chamber was transferred to a new 24-well transwell plate, which the lower chamber loading 4% paraformaldehyde to fix the cells. After 30 min, chamber was washed softly 3 times with PBS and stained with crystal violet (0.1%, Beyotime, China) for 30 min. At last, the cells in 5 random areas were counted and photographed by high definition camera microscope. All the experiments were repeated three times.
Cell adhesion assay
In cell adhesion assay, 24 well plates were coated with the rat tail type I collagen (C8062, Solarbio, China), washed with PBS 2–3 times, and then placed at 37 ℃ overnight. The treated cell suspension (about 50000 cells) was planted on the coated culture plate and placed in the 37 ℃ cell incubator for 30 minutes and 1 hour respectively. The liquid was removed and washed with PBS for 1–2 times. First, the cells were fixed with 4% paraformaldehyde solution for 20 minutes, washed gently for 1–2 times, and then added crystal violet staining solution, dyed for 20 minutes, gently washed 1–2 times, and finally taken photos in 3 areas randomly selected under the microscope.
The treated GC cells which were cultured on 10 cm culture plates in the logarithmic growth phase were digested, suspended in PBS. Then the cells were injected into the nude mice intraperitoneally at the standard of 1×105 cells/100µl per nude mice. After the injection, rubbing the abdomen of the nude mice to make the cells evenly to distribute in the abdominal cavity. All 6-week-old male nude mice were divided into 4 groups: NC group and CPXM1 overexpression group, shNC group and shCPXM1 knockdown group (5 mice in each group). Nude mice were sacrificed 6 weeks after operation, and the number of tumor nodules in the abdominal cavity and liver surface nodules were counted.
RNA extraction and quantitative real-time polymerase chain reaction (RT-qPCR)
Total RNA was extracted using TRNZol Universal Reagent (TIANGEN, China) according to the manufacturer's instruction. Total RNA (1µg) was reverse-tanscribed using FastKing RT Kit with gDNase (TIANGEN, China). The primers for GAPDH, CPXM1 were:
qRT-PCR was performed using SuperReal PreMix Plus (TIANGEN, China) on QuantStuido 3 Real time PCR instrument (Thermo Fisher Scientific, USA). The reaction program was set to the following parameters: 95 ℃ for 15 min; 40 cycles of 95 ℃ for 10 s, 60 ℃ for 30 s, 72 ℃ for 30 s; one cycle of 95 ℃ for 15 s, 60 ℃ for 1 min.
Protein extraction and western blotting
50mg fresh tissue was cleaned with cold phosphate-buffered saline (PBS), cut into small pieces and lysed in 500 µl radioimmunoprecipitation assay (RIPA) lysis buffer (50mM Tris pH 7.4, 150mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, EDTA; Beyotime, China) with phenylmethane sulfonyl fluoride (PMSF; Beyotime, China) by homogenizer. 6-well plates seeded cell were cleaned with cold PBS and lysed in 150 µl RIPA lysis buffer with PMSF. Tissue or cell lysate were centrifuged at 12,000 rpm for 5 min at 4 ℃ and the total protein was collected in the supernatant. When the total protein of cell supernatant was extracted, the cells were firstly seeded in 10 cm dishes with complete media. Then, the complete media was replaced by 10 ml RPMI-1640 without PBS and the cells cultured 48 h. Finally, the cell supernatant was collected into Amicon Ultra-15 10K device and centrifuged at 4000 g for 1 h at 4 ℃. All extracted protein was measured the protein concentration by bicinchoninic acid (BCA) protein assay regent kit (Beyotime, China) for subsequent western blotting. 15–20 µg protein sample loaded into per lane were separated by 8% SDS-PAGE (Beyotime, China). After electrophoresis for 1 h 30 min, the protein was transferred to nitrocellulose membranes (Pall, Mexico), which were subsequently blocked with 5% bovine serum albumin (BSA; Biofroxx, Germany) for 1 h at room temperature. The blocked nitrocellulose membranes were incubated in the primary antibodies at 1:1000 dilution at 4℃ overnight and then incubated with secondary antibodies at 1:5000 dilution for 1 h at room temperature. After each antibodies incubation, the membranes were washed three times using triethanolamine buffered saline with 0.1% Tween-20 (TBST) for 15 min. Finally, the ECL buffer was used to detect bolts by the X-ray film. The following primary antibodies were used: anti-CPXM1 (GTX119190, 1:1000, GeneTex, ), anti-GAPDH (AF7021, 1:5000, Affinity, China), anti-ITGB2 (AF6399, 1:1000, Beyotime, China), anti-FAK (D5O7U, 1:1000, CST, USA), anti-pFAK (D20B1, 1:1000, CST, USA), anti-Src (32G6, 1:1000, CST, USA), anti-pSrc (D49G4, 1:1000, CST, USA), anti-Erk (137F5, 1:1000, CST, USA), anti-pErk (197G2, 1:1000, CST, USA), anti-MMP2 (40994, 1:1000, CST, USA), anti-MMP9 (13667, 1:1000, CST, USA).
The treated cells were cultured on the slide for 24 hours to make the density reach 50% confluence. The culture medium was aspirated and washed with PBS which was preheated at 37 ℃ for 1–2 times. We used 4% formaldehyde solution in PBS for cell fixation, and fixed for 10 minutes at room temperature. It should be noted that avoid the fixative containing methanol components, because methanol may destroy actin during the fixation process. At room temperature, the cells were washed 2–3 times with PBS for 10 minutes each time, permeabilized with 0.5% Triton X-100 solution for 5 minutes, and then washed 2–3 times with PBS for 10 minutes each time. The cells were covered 200 µl of the prepared rhodamine-labeled phalloidin working solution, and incubated for 30 minutes at room temperature in the dark. It should be noted that in order to reduce the background, 1% BSA can be added to the rhodamine-labeled phalloidin working solution. In order to avoid volatilization of the solution during the incubation, the coverslip can be transferred to a sealed container. After the incubation, the slides were washed with PBS 3 times for 5 minutes each time. We Used 200µl of anti-fluorescence quencher (including DAPI) solution to counterstain the cell nucleus for about 30 seconds, washed the slides with PBS, and then placed it upside down on the coverslips which had been dripped with a drop of water-soluble mounting tablets. The excess mounting tablets was removed. The fluorescence microscope or confocal microscope, selected TRITC excitation/emission filter (Ex/Em = 540/570nm) and DAPI excitation/emission filter (Ex/Em = 364/454nm), was used to take pictures which selected randomly 3 areas.
Each experiments were repeated three times, and the results were analyzed by Student’s t-test. All p values < 0.05 were considered to be statistically significant. R 4.0.2, Graphpad Prism 8.0.2, SPSS 22, Image J and other softwares were used for data processing, analysis and mapping.