2.1 GEPIA (Gene Expression Profiling Interactive Analysis) dataset
GEPIA (http://gepia.cancer-pku.cn/) is an online data analysis website for analyzing RNA-sequencing expression data of 9736 tumors and 8587 normal samples from TCGA and GTEx databases. The expression differences of GRWD1 amid cancer tissues and adjacent tissues of gastric cancer (GC) patients were studied using GEPIA. The correlation between GRWD1 gene and TACE, Notch-1, HES-1, and other genes in Notch signaling pathway was examined through GEPIA.
2.2 Tissue samples and clinical data
A total of 99 paraffin sections of GC tissues from patients in the First Affiliated Hospital of Anhui Medical University from 2015 to 2016 (matched paracancerous tissues were reserved from a section of resection specimens > 5 cm from the tumor) were used for immunohistochemical staining. Fresh GC tissues and corresponding paracancerous tissues were taken from hospitalized patients in 2021. All tissues were surgically removed, and the patient had not received chemotherapy or radiation therapy prior to surgery. The Medical Ethics Committee of the First Affiliated Hospital of Anhui Medical University approved this study following the ethical guidelines of the Declaration of Helsinki.
2.3 IHC
The surgically resected tumor specimens were fixed with 10% neutral formalin, embedded in paraffin, and cut into 4 μm sections. Sections were dewaxed in xylene, rehydrated in graded alcohol series, and autoclaved for 3 min with 0.01 mol/L citric acid buffer. Endogenous peroxidase activity was blocked by 0.3% hydrogen peroxide. Sections were incubated with normal goat serum (5%) for 30 min at 20 °C to reduce nonspecific binding. After blocking with goat serum for 1 h, it was incubated with anti-GRWD1 antibody (1:500 dilution, HPA042643, Sigma, USA) overnight at 4 °C. Positive signals were visualized by DAB kit (Sevier Bio, Wuhan, China) and then scored. Scores were made according to the percentage of positive cells and staining intensity in GRWD1. Staining intensity score: -, 10%, +, 10% - 25%, ++, 26% - 50%, +++, > 50%. All scores were divided into two categories: GRWD1 high expression (++, +++) and low expression (-, +). Scoring was done by two independent people to avoid subjectivity.
2.4 Cell culture and transfection
Human GC cell lines BGC-823 and MGC-803 were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum and 100 U/mL penicillin at 37 °C, 5% CO2. The cell transfection was performed with Lipofectamine 3000 reagent. In GRWD1 gene knockout experiment, GRWD1-specific small interfering RNA (sense:5′-CCAGAGCAACAGACUGAUGAUTT-3′, antisense: 5′-AUCAUCAGUC UGUUGCUCUGGTT-3′) or interference control si-NC (Shanghai Shenggong) was introduced into cells for 48 h. PCR-amplified human GRWD1 cds region was cloned into a pLVX-puro vector to construct a GRWD1 cDNA recombinant expression vector. HEK293T cells were transfected with lentiviral vectors. The medium was changed after 8 h, and the supernatant was collected and filtered after 48 h. BGC-823 and MGC-803 cells were infected with viral supernatant and screened with 1 µg/mL Puromycin for 5 d.
2.5 Western blot analysis
The cells were harvested, lysed with ice-cold lysis buffer, the extract was centrifuged, and the supernatant was diluted 1:4 with protein loading buffer. 10 μg of protein was separated by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and transferred to PVDF membrane. After blocking with 5% non-fat milk for 1 h at room temperature, membranes were incubated with primary antibodies overnight at 4 °C. After the membrane was washed three times with PBST, it was incubated with a horseradish peroxidase-labeled secondary antibody for 1 h at room temperature. Finally, the signal was detected using an enhanced chemiluminescence system. GRWD1 was purchased from abbexa (Cat. #: abx036119 Rabbit 1:1000), GAPDH (Cat. #: R24403 Rabbit 1:2000), Notch-1 (Cat. #: 380355 Rabbit 1:1000), HES-1 (Cat. #: 381205 Rabbit 1:1000), TACE (Cat. #: 381683 Rabbit 1:1000), RBPJK (Cat. #: 382329 Rabbit 1:1000) were purchased from Chengdu Zhengneng Biotechnology Co., Ltd.
2.6 Cell proliferation and colony formation assays
The cells in the logarithmic growth phase were taken, trypsinized, and seeded into 96-well plates in the cell proliferation experiment. The amount of cell suspension in each well of the 96-well plate was 100 μL, the number of cells in each well was 5000, and each group was set up with three replicate wells, which were cultured in an incubator for 24h, 48h, and 72 h, respectively. After incubation, the medium was discarded, CCK8 reagent was added to incubate for 1-2 h, and the absorbance was measured at 450 nm with a microplate reader.
In colony formation experiments, the cells were seeded in 6-well plates at a density of 500 cells/dish and cultured for 10-15 days. The cells were washed with PBS and stained with crystal violet, and colonies of more than 50 cells were counted. At least three independent experiments were performed under the same conditions.
2.7 cell invasion assay
Cell migration assays were performed in 24-well Transwell chambers with a pore size of 8 μm. After 24 hours, all cells were trypsinized and 10 x 104 cells were transferred to the upper perforated chamber in 200 μl and 5% fetal bovine serum, while 600 μl and 20% calf serum were added to the lower perforated chamber. After 24 h of culture, the cells on the membrane surface were removed with cotton swabs, fixed with formaldehyde, and stained with crystal violet. Ten regions were randomly selected under high magnification to count the number of transitional cells. This experiment was repeated three times.
2.8 Wound Healing Assay
After transfection, BGC-823 or MGC-803 cells were seeded on 6-well plates, an artificial scratch wound was formed with a 20 µl pipette tip, washed 3 times with PBS, and the detached cells were taken out. The percentage of serum used for wound healing assays was 0.5%. After 48h or 72h incubation, images of cell migration were captured with an inverted microscope and evaluated by measuring the difference in wound width.
2.9 Animal model
One-month-old BALB/c nude mice were purchased from the Animal Center of Anhui Medical University School of Medicine and divided into three groups of six mice each. Two groups were inoculated with pLVX-GRWD1 MGC-803 cells, and one group was administered with DAPT at a dose of 10 mg/kg/day through the intragastric route(Kalantari E et al.2013),the other group was given DMSO at a dose of 10 mg/kg/day via intragastric route. The remaining group was inoculated with pLVX-vector MGC-803 cells and administered DMSO by gavage at 10 mg/kg/day. The inoculation was done by subcutaneous injection of 5×106 GC cells in the right armpit. The tumor volume was measured every five days, and the total tumor volume was calculated as V (volume, mm) = 0.5 × L (length, mm) × W2 (width, mm). Eight days after inoculation, the mice were gavaged and sacrificed five weeks later. The subcutaneous tumors were excised and weighed. The care of experimental animals and animal experiments were in accordance with animal ethics guidelines and approved by the Experimental Animal Ethics Committee of Anhui Medical University. The permit number of the approved animal work provided by the ethics committee was LLSC20211261.
2.10 Statistical Analysis
All data were analyzed using SPSS 16.0 statistical software (SPSS Inc., Chicago, IL, USA). Survival rates of all patients were tested using the Kaplan-Meier method for long columns. Differences between the two groups were compared using a t-test. The relationship between its expression and clinical features was analyzed with Χ2. P < 0.05 indicates that the difference was statistically significant.