2.1. Bioinformatic Analysis
NCBI-Gene Expression Omnibus (GEO) (https://www.ncbi.nlm.nih.gov/) is a public resource that includes microarray and high throughput sequencing data. A total of three microarray datasets have been selected and retrieved (GSE49051, GSE54129, and GSE112369). As a primary objective, wesearched all datasets for gene expression patterns specific to GC cells compared to their normal neighboring cells. To identify clinical characteristics linked to stomach cancer, the cancer genome atlas (TCGA) database was employed.Our analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) was conducted after integrating all the expression profiles. For gene set enrichment analysis (GSEA), PLCD3 coexpressed genes were selected under the following conditions: enrichment analysis, KEGG pathway, rank criteria, meta-P-value, and minimum number of genes. We utilized the limma package in R to identify genes with distinct expression,using the cut-off criteria of |Fold change| >0 and P-Value < 0.05.
2.2. Patients and GC specimens
Between July 2015 and May 2017, we collected 80 pairs of formalin-fixed and paraffin-embedded humanGC specimens ranging in age from 43 to 77 years old. In addition, 16 pairs of freshly extracted GC tissues were taken from nine men and seven women aged 46 to 67. In this study Northern Jiangsu People's Hospital, School of Clinical Medicine, Yangzhou University (Yangzhou, China) provided the specimens. Notably, none of the patients had preoperative chemotherapy. Two distinguished pathologists verified stomach cancer diagnosis. The American Joint Committee on Cancer TNM staging approach for gastric cancer (AJCC-8 TNM) was utilized to assess the disease's stage. Northern Jiangsu People's Hospital, YangzhouUniversity's School of Clinical Medicine, and the Ethics Committee (Yangzhou, China) have all approved this project, with the permission number 2019 KY-022. Prior to the trial, all subjects provided written informed consent.
2.3. Immunohistochemistry (IHC)
GC samples were fixed in paraffin and cut into 4 mm thick tissue slices. After that, the sections were fixed for one day in a 10% formalin solution before being placed on slides. Theslides were baked in an oven at 60°C for two hours on the first day, and then allowed to cool to room temperature. In order to remove the wax, the tissue was treated with xylene and ethanol concentrations ranging from 100–50%. In the next step, sodium citrate (Cat#C1032, Solarbio, Beijing, China) was used for 30 minutes in order to repair the antigen. In order to block endogenous peroxidase activity, 3% H2O2 (PV-9000, OriGene, Jiangsu, China) was added. After treating the sample with goat serum (PV-9000, OriGene, Jiangsu, China) for 30 minutes, the primary antibody PLCD3 (1:50, PK83857, Abmart) wasadded dropwise. A secondary antibody (PV-9000, OriGene, Jiangsu, China) was added dropwise on the second day and incubated for 30 minutes at 37°C. For nuclear staining, 1:20 DAB chromogenic solution (ZLI-9018, OriGene, Jiangsu, China) was used, followed by hematoxylin (Cat.C0107-100ml, Beyotime, Shanghai, China). In order to photograph the blocking, an Olympus BX53 fluorescence microscope was used.
2.4. Cell lines and cell culture
We obtained the normal human gastric cell line (GES-1) and five human GC cell lines (AGS, MKN45, BGC-823, N87, HGC-27) from the Cell Bank of the Chinese Academy of Sciences, Shanghai Institute ofCell Biology, Chinese Academy of Sciences, Typical Culture Repository. The cells were cultured in RPMI1640 medium (Cat. No. 31800, Solarbio, Beijing, China). The cells were cultivated at 37°C in a 5% CO2 incubator (Thermo Scientific, USA) according to the manufacturer's instructions. As a supplement to the medium, 10% fetal bovine serum (Lot.NM220304, Eallbio, Beijing, China), 100 units of penicillin and100 units of streptomycin (60162ES76, Yeasen, Shanghai, China) were added. There was no sign of mycoplasma or any other fungi in any of the cell lines used in the experiment.
2.5. Plasmid Infection and Transient Transfection
GenePharma (Shanghai, China) constructed the small interfering PLCD3 (si-PLCD3) and negative control small interfering (siCtrl). The sequences are as follows: siCtrl (forward: 5′-UUCUCCGAACGUGUCACGUTT-3′, reverse: 5′-ACGUGACACGUUCGGAGAATT-3′); si1-PLCD3 (forward: 5′-GCAGCUCAUUCAGACCUAUTT-3′, reverse: 5′-AUAGGUCUGAAUGAGCUGCTT-3′); si2-PLCD3 (forward: 5′-GCCCACUACUUCAUCUCUUTT-3′, reverse: 5′-AAGAGAUGAAGUAGUGGGCTT-3′). A pcDNA-PLCD3 construct was cloned into the BamH1/EcoRI restriction digest site of the pcDNA3.1 plasmid by GenePharma. The primer sequence for PLCD3 with the BamH 1/EcoRI enzyme site is as follows: oe-PLCD3 (forward: 5′-GCTTGGTACCGAGCTCGGATCCGCCACCATGCTGTGCGGCC-3′, reverse: 5′-TGCTGGATATCTGCAGAATTCTCAGGAGCGCTGGATGCGGATTTGGATGA-3′). 2×105 GC cells were inoculated into six-well plates and grown overnight. The plasmid was then infected and transiently transfected with lipofectamine 2000 (Lot.2357812, Invitrogen, USA) the next day. Transfected cells were incubated at 37°C in a 5% CO2 incubator for 48 hours.
2.6. Real-time quantitative PCR(RT-qPCR) assays
The total RNA was extracted from the cell lines using Trizol reagent (R701-01-AA, Vazyme, Nanjing, China). We transcribed the extracted RNA into cDNA using a reverse transcription kit (017E2282IA, Vazyme, Nanjing, China) and performed RT-qPCR using a SYBR Green PCR Kit kit (Cat:11203ES08, Yeasen,Shanghai, China) as described in a previous study16. Using StepOne Software v2.3, Thermo Fisher Scientific, USA, the amplification procedure was a two-step process involving 95°C for 5 minutes, 95°C for 10 seconds, and 60°C for 30 seconds, with a cycle count of 40. Primers were provided by Sangon Biotech, Shanghai, China, and their sequences were as follows: PLCD3-forward (5'-CATTCGGGAGGCAGGGAAC-3'), PLCD3-reverse (5'-TTCCACATCTCCTGGGGACT-3'). GAPDH primer sequences are as follows: GAPDH forward (5′-TGACATCAAGAAGGTGGTGAAGCAG-3′) and GAPDH reverse (5′-GTGTCGCTGTTGAAGTCAGAGGAG-3′). We used the 2−ΔCt method using GAPDH as an internal reference to determine the relative expression of the targets. RedSafe nucleic acid staining solution was used to visualize PCRproducts after electrophoresis on 1% agarose gels.
2.7. Western blot
Cells were lysed with RIPA lysis solution (Cat# R0010, Solarbio, Beijing, China) at 4°C. A lysate of scraped cells was collected, centrifuged at 14,000 rpm for 15 minutes at 4°C, and the supernatant was removed. The absorbance values of the cells were measured at 570 nm using the BCA kit (No.927E2220KA,Vazyme, Nanjing, China). The samples were boiled for 10 minutes after being mixed with 5X Loading buffer. Afterwards, they were electrophoresed on an SDS-PAGE gel and electrotransferred to a PVDF membrane (Lot: 0000161247, Immobilon-P, Merck Millipore, Ireland). Closure with 5% skimmed milk at room temperature for 90 minutes, followed by overnight shaking at 4°C with the primary antibody. The following antibodies were included in the study: PLCD3 (1:1000, Abmart), GAPDH Mouse mAb (1:10000, abclonal), BAX (1:1000, ZENBIO), P53 (1:1000, ZENBIO), BCL2 (1:1000, ZENBIO), MDM2 (1:1000, Cell Signaling Technology), CDK2 (1:1000, ZENBIO), CDK6 (1:1000, ZENBIO), MTOR (1:1000, ZENBIO), P-MTOR (1:1000, ZENBIO), MMP2 (1:1000, Cell Signaling Technology), MMP9 (1:1000, Cell Signaling Technology), JAK2(1:1000, Cell Signaling Technology), P-JAK2 (1:1000, Cell Signaling Technology), STAT3 (1:1000, Cell Signaling Technology), P-STAT3 (1:2000, Cell Signaling Technology), N-Cadherin (1:1000, Affinity), E-Cadhein (1:1000, Affinity). The following day, secondary antibodies Goat anti-Rabbit IgG (1:8000, ZENBIO) and Goat anti-Mouse IgG (1:8000, ZENBIO) were incubated at room temperature for 1h, followed by development with ECL Immunoblot Detection Reagent (Cat.#KF8003, Affinity, USA).
2.8. Cell Counting Kit‑8 (CCK‑8) assay
In each well of a 96-well plate (Lot:111622BL01, NEST, Wuxi, China), 1000 cells were inoculated and incubated overnight at 37°C. The CCK-8 reagent (Lot#255919, MedChemExpress, USA) was added at 24 hours, 48 hours, 72 hours, and 96 hours following the addition of the CCK-8 reagent. After 2 hours of incubation, the absorbance value of each well was measured using a microplate reader (Thermo Scientific, USA).
2.9. Colony formation assay
In six-well plates, 600 cells were spread per well after small interferences and plasmid infection. The cells were washed three times with PBS after two weeks of culture, then fixed in 4% paraformaldehyde (Lot22360481, Biosharp, Beijing, China), and stained with 5% crystalline violet solution (Cat# G1075, Solarbio, Beijing, China). Imagej (National Institutes of Health, Bethesda, USA) software was used to image and count the cell colonies as described in previous studies.
2.10. Wound healing assay
The cells were inoculated into 6-well plates (Corning, USA) and cultured for one day in serum-free RPMI-1640 medium. After the cell density reaches 80–90%, the monolayer is scraped vertically downwards using a yellow tip with a range of 200 mL and the residual cells are washed with PBS. After that, the medium was changed to 2% FBS. At 0h, 24h, and 48h, the cells were observed under a microscope (OLYMPUS CKX53, France) and the migration phenomenon was recorded.
2.11. Transwell migration and invasion assays
The migration experiments were conducted using small chambers (353095, Corning, USA) in 24-well plates (Lot:061722BH01, NEST, Wuxi, China). In the small chamber, the upper chamber is filled with 200 ml of RPMI-1640 cell suspension without FBS (number of cells: 1×104) and the lower chamber is filledwith 500 ml of RPMI-1640 medium containing 10% FBS. For invasion experiments, 10% artificial substrate gel (cat. no.356234, BD Biosciences, USA) was spread in the upper chamber of the small chamber the night before the experiment, 200 liters of FBS-free medium was added to the upper chamber and 500 liters of complete medium was added to the lower chamber. They were fixed with 4% paraformaldehyde for 24 hours before dyeing with 5% crystalline violet solution. A light microscope was used to capture the images, and five randomly selected fields of view were analyzed statistically.
2.12. Immunofluorescence staining
N87, HGC-27, AGS, and BGC-823 cell lines were transfected the night before and plated in confocal culture dishes (Cat. No.: 801001, NEST, Wuxi, China). The cells were rinsed three times in PBS the next day before being fixed in 4% paraformaldehyde for 15 minutes at room temperature. They were then permeabilized with 0.1% Triton X 100 for 3 minutes. The cells were blocked with normal goat serum (Cat.No. SLO38, Solarbio, Beijing, China) for 30 minutes at room temperature and then incubated with diluted primary antibody PLCD3 (1:100, Bioss, Beijing, China) overnight at 4°C. A fluorescent secondary antibody, Goat Anti-Rabbit IgG (H + L) Fluor594-conjugated (1:100, Affinity, USA), was added dropwise in adark room, incubated for 1 hour, and stained with DAPI (C1005, Beyotime, Shanghai, China) for 10 minutes the following day. The images were finally acquired by observation with a laser confocal microscope (Carl Zeiss LSM710, Germany) after four washes with PBS.
2.13. Statistical analysis
Statistical analysis was performed using GraphPad Prism 9 (San Diego, CA, USA). The Student's t-test was used to compare differences between the two groups. ANOVA was used for groups of three or more. Kaplan-Meier survival curves and the log-rank sum test were used to analyze the survival curves. According to the following criteria, statistical significance was determined: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.