Tissue microarray and clinical samples
Tissue microarray (G6038-3) including 48-paired GC tissues and relevant adjacent non-neoplastic tissues were obtained from Servicebio Biotech (Wuhan, China) containing clinical information such as gender, age, tumor location and size, metastasis, vessel carcinoma embolus and the overall survival of patients. After immunohistochemistry assay, the immunostaining intensity scores were evaluated by two experienced pathologists, performed double-blind reading. The percentage of positive cells and the staining intensity were analyzed by semi-quantitative results according to the following criterion: On the one hand, the percentage of positive cells was divided into 5 grades: the number of positive cells < 5% was 0, 5% ~ 25% was 1, 26% ~ 50% was 2, 51% ~ 75% was 3, 76% ~100% was 4 points. On the other hand, the staining intensity was recognized as 4 grades: negative was 0, weak was 1, moderate was 2, and strong was 3 points. The total scores were calculated by adding up the product on a scale range of 0-12. Tissues of total score < 7 were classified into low expression while score ≥ 7 were recognized as high expression. 33 pairs of clinical tissues were collected from the Second Affiliated Hospital of Xi 'an Jiaotong University with informed consent from patients.
Immunohistochemistry (IHC)
Then tissue microarray and paraffin sections were soaked in xylene twice for deparaffinating. Rehydrated the slides with series ethanol (100%, 95%, 85% and 75%). Slides were evenly spaced in citrate buffer for microwaving treatment to perform antigen retrieval. Afterwards, added 3% hydrogen peroxide to remove endogenous peroxidase and then the slides were blocked with goat serum for 30 minutes at room temperature (RT). Then, added appropriate concentration of primary antibodies and incubated at 4°C overnight. The next day, added secondary biotinylated antibodies and incubated at RT for 1 hour. Then, fresh DAB staining solution was added and the staining degree was observed under a microscope. Counterstained in haematoxylin bath for 30-60 seconds. Finally, dehydrated the slides in graded ethanol (75%, 85%, 95% and 100%) and transferred to dimethylbenzene, and then added neutral resins to seal the slides.
RNA extraction and qRT-PCR
Total RNA was extracted by TRIzol (Invitrogen, Thermo, Waltham, MA, USA). Afterwards, the messenger RNA was transcribed reversely by the Transcriptor First-Strand cDNA Synthesis Kit (Roche, Basel, Switzerland). Subsequently, we used the FastStart Universal SYBR Green Master (Roche) to conduct qRT-PCR analysis following the manufacturer’s instructions. All of the primer sequences were listed in Additional file 4: Table S2.
Cell culture
Human GC cell lines HGC27, BGC823, MKN45, SNU-1 and the normal gastric epithelial cell line GES1 were purchased from Genechem (Shanghai, China), identified by STR profiling and tested free of mycoplasma contamination. All cell lines were cultured in high glucose DMEM (HyClone, Logan, Utah, USA) supplemented with 10% fetal bovine serum (Gemini, Calabasas, CA, USA) and 1% penicillin-streptomycin (Gibco, Grand Island, NY, USA) in a humidified incubator in 5% CO2 at 37°C.
Western blotting
Total protein was extracted using cold RIPA cracker buffer (Beyotime, Shanghai, China). Cytoplasmic and nuclear extracts were separated by applying NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific™, MA, USA). Determine the protein concentration of each cell lysate using BCA Protein Assay Kit (TIANGEN, Beijing, China). Next, the same amount of protein samples was applied to SDS-PAGE gel wells and then transferred to the PVDF membranes. The membrane was incubated overnight at 4 ℃ in a primary antibody solution. The involved antibodies were demonstrated in Additional file 5: Table S3. The next day, the membrane was incubated with horseradish peroxidase bound secondary antibody and visualized with enhanced chemiluminescence reagents.
Lentivirus and siRNA transfection
The overexpression lentivirus and control lentivirus were obtained from Hanbio Biotrchnology (Shanghai, China). The component order of overexpression lentivirus was HBLV-h-CARM1-3xflag-ZsGreen-PURO, while the control component was HBLV-ZsGreen-PURO. The transduction was performed according to the manufacturer’s protocol. Then culture the transfected cells in medium containing 2.5 µg/mL puromycin to obtain stable overexpression cells. HGC27 and BGC823 CARM1-overexpression cells were transfected with specific siRNAs (GenePharma, Shanghai, China), targeting CARM1 and TFE3 respectively. Lipofectamine 2000 reagent (Invitrogen, Calrsbad, CA) was added to facilitate transfection in accordance with the manufacturer’s instructions. The interference sequences involved were shown in Additional file 4: Table S2.
Transmission electron microscopy
Cells were harvested and fixed at 4℃ for 2-4hours with electron microscope fixation solution (Servicebio, China) and further fixed with 1% osmium ·0.1M phosphate buffer for 2 hours. Dehydrated cells in graded ethanol (50%, 70%, 80%, 90%, 95% and 100%) and 100% acetone twice. Embed and cut cells into slices of 60-80nm with ultra-thin slicing machine. Then, double staining of 2% uranium acetate-lead citrate was used. At last, under transmission electron microscopy (HITACHI, Tokyo, Japan), photographs of eight random fields in every sample were captured.
Immunofluorescence
Cells were cultured on small cover glass. 4% paraformaldehyde was added into the hole for cell fixation for 30 minutes at RT. Then treat cells with 0.1% Triton 100 to permeate cells for 15 minutes. Block cells with 5% goat serum without washing. Then sufficient amount of the appropriate concentration of primary antibodies were added overnight in a moist container at 4℃. In the following day, cells were incubated with fluorescence labeled secondary antibody and for 1 hour and DAPI for 1 minute in dark. In the end, sealed the coverslip with anti-fading buffer (BD Biosciences, NJ, USA). Photographs of five random fields were captured with confocal microscopy (Nikon C2, Tokyo, Japan).
CCK8 assay
Cell suspensions were added in 96-well plates with 3 × 103 cells per well in 100 µL DMEM and incubated for 1-4 days. Each well was added 10ul CCK-8 regent (7 Sea Pharmatech Co., Ltd, Shanghai, China) and incubated in the cell incubator for 1 hour. Then read the absorbance value at 450nm determined by a microplate spectrophotometer (Thermo, Waltham, MA, USA).
Colony formation assay
Add cells in the exponential growth phase at a density of 1 × 103 cells each well in 6-well plates. Then cultivate the cells for 10-14 days until visible clones were observed. In the process, replace fresh culture medium timely according to the pH change. Afterwards, fixed cells with 4% paraformaldehyde for 20 minutes and stained with crystal violet dye for 10 minutes. Finally, counted the number of colonies containing more than 50 cells.
Flow cytometry
2 × 105 cells were plated in 6-well plates and grew in serum-free medium for 24 hours to keep the same cell cycle. Then culture cells for another 24 hours. The adherent cells were digested and fixed by 70% ice ethanol at 4℃ overnight. The next day, the fixed cells were washed and resuspended with 500µL PI/RNase Staining Buffer (BD Biosciences, Franklin Lakes, NJ, USA). After culturing for 15 minutes in dark at RT, flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA) was used to detect different cell cycle phases of cells.
Seed 1.5 × 105 cells in 6-well plates each well and then continue to cultivate cells to a confluence of 80%-90%. Afterwards, the proportion of apoptosis cells was determined by PE Annexin V/7-amino‐actinomycin (7-AAD) Detection Kit (BD Biosciences, NJ, USA) following the manufacturer’s instructions and analyzed by flow cytometry.
In vivo xenograft tumor model
A subcutaneous xenograft model was established using 5-6 weeks old male BALB/c nude mice (Xi'an Jiaotong University Medical Laboratory Animal Center, Xi'an, China). The experiments were approved by the animal ethics committee of Xi'an Jiaotong University. We subcutaneously injected 1.0 × 106 tumor cells into the left groin of nude mice where blood flow was abundant. The volume of subcutaneous tumor was evaluated every 3 days. For the drug research, nude mice injected with CARM1 overexpression cells were randomly divided into four groups at day 6 when tumor volumes reached 50 mm3. CARM1 inhibitor EZM2302 was performed twice a day at 100 mg/kg i.p. HCQ was administered once a day at 50 mg/kg i.p. The combination group was given two treatments and the control group was only intraperitoneally injected with PBS. When the subcutaneous tumor showed ulceration and necrosis, the mice were sacrificed, the transplanted tumors were removed, and the weight and volume were measured.
Immunoprecipitation assay
The cells were harvested and added appropriate amount of IP lysis buffer (including protease inhibitor) for 30 min at 4°. After centrifugation, 6 µg CARM1 antibody or control IgG and 40 µ L Protein A/G Mix Magnetic Beads (Merck Millipore, Germany) were added into the supernatant and incubated at 4°C overnight. After immunoprecipitation reaction, the protein was carefully eluted from the protein A/G-beads and denatured for western blotting analysis.
Statistical analysis
We used GraphPad Prism 6 (San Diego, CA, USA) and SPSS 20.0 (Chicago, IL, USA) to perform statistical analysis. When comparing the differences in measurement data between the two groups, Student’s t test was used. When comparing the differences between more than two groups, ANOVA was applied, in which p value were adjusted in face of multiple comparisons. Before Student’s t test and ANOVA, a variance homogeneity test and normality analysis were carried out. Overall survival curves were analyzed by the Kaplan–Meier method and log-rank tests. Chi-squared test was utilized to estimate the difference of CARM1 level between gastric cancer tissues and adjacent non-tumor tissues. The hazard ratio in clinical samples was determined by cox proportional hazards model for univariate and multivariate analyses. All values demonstrate means ± SD. All statistical tests were two‐sided, and P < 0.05 was considered statistically significant.