Patient recruitment and sample collection
This study was approved by the Ethics Committee of Quanzhou First Hospital Affiliated to Fujian Medical University and informed consent was obtained from the patients. Patients who were diagnosed with gastric cancer in our clinical center from Feb, 2019 to Aug, 2020 were retrospectively enrolled in this study. No prior surgical treatment, radiotherapy or chemo-therapy were conducted. Clinical samples of gastric cancer tissues and adjacent normal tissues were gathered post-surgical treatment. Samples were stored in liquid nitrogen for subsequent experiments.
RNA extraction and qRT-PCR experiments
Total RNA samples from cell line samples and clinical tissue were extracted by RNAiso Plus agent (TaKaRa, Dalian, China) according to the standardized protocol. The level of RNA was quantified and reverse transcription polymerase chain reaction was performed to generate cDNAs. The levels of mRNA expression were quantified by real-time PCR qRT-PCR reaction with condition setting as 94 °C for 30 s, 55 °C for 30 s, 72 °C for 90 s, with total cycles of 40. Primers used in this study were listed in detail (Additional file 1: Table S1).
Cell line culturing
Gastric cancer cell lines (MKN45, HGC27, MGC-803, BGC-823, AGS, MKN28) and human normal gastric epithelial cell (GES-1) were purchased from American Type Culture Collection (ATCC; Manassas, VA, USA). RPMI-1640 medium with 10% fetal bovine serum (FBS; Hyclone, South Logan, UT, USA). 100 IU/mL penicillin with 100 µg/mL streptomycin (Invitrogen, Carlsbad, CA, USA) were applied for cell culture, under 37 °C, with 5 % CO2.
Lentiviral transfection
pLVX lentiviral vector was purchased from Clontech (USA). Polymerase chain reaction (PCR) was conducted using templates of IGHG1 specific shRNA and full length of IGHG1 cDNA sequences. PCR products were firstly purified using 1 % agarose gel and double digested by BamHI and EcoRI along with empty pLVX vectors. Ligation reaction was performed overnight between vector and the purified PCR products by T4DNA ligase. Ligation product was further used for the E.coli DH5α competent cells transformation. Cell clones were subsequently placed into the ampicillinum containing-LB plate and were incubated overnight at 37 °C. Positive clones were gathered and the plasmid was extracted and sequenced (Shanghai Invitrogen Biotech Co., Ltd). Afterwards, the lentiviral vectors carrying the IGHG1 shRNA / cDNA and control vectors were packaged and subsequently added into gastric cancer tumor cell groups with multiplicity of infection (MOI) of 20 (control group with empty vectors, overexpression group with IGHG1 cDNA vectors, suppressed group with IGHG1 shRNA vectors), and the vectors titer was set as 1 × 109/mL. Then the fluorescent protein expression level was detected and transfection efficiency was evaluated 24–48 h post-transfection.
Western blot
2 × 106 cells per cell group were washed twice with cold PBS, re-suspended and treated by ice-cold cell lysis buffer RIPA agent (Beyotime, Shanghai, China) to extract total protein from the samples and BCA protein assay kit was used for quantification. Firstly, protein samples were separated by SDS-PAGE and 10 % separating gels and then samples were electroblotted onto PVDF membranes (Immobilon-P; Millipore, Billerica, MA). After blocking in Tris buffer (50 mM Tris, pH 7.5) containing 5 % skim milk, the membranes were incubated overnight at 4 °C with primary antibodies. Secondly, after rinsing with the Tris-Buffered Salin and Tween buffer solution (TBST; Sigma-Aldrich, St. Louis, MO, USA), they were then incubated with the secondary antibody for 2 h. Chemiluminescence was used to expose the protein bands on the membrane.
Cellular proliferation assay
For CCK8 assay, each group of cells were seeded with density (3000 cells/well) into 96-well plates. After vector transfection, the cells were incubated for 72 h. 10 µL CCK8 was added to each well and incubated for 4 h at 37 °C. Microplate reader scanning was conducted at 450 nm to quantify cellular proliferative status. Cell survival rates were measured at three time points of the cell growth curve through the log phase of growth for each cell line.
For Edu assay, experiment was conducted according to the protocol of BeyoClick™ EdU Cell Proliferation Kit (Beyotime, Shanghai, China). Diluted EdU medium was added into samples and incubated for 2 h, and then washed in PBS and subjected to DAPI staining. All groups of samples were then inspected by inverted microscope (Olympus, Tokyo, Japan).
For spherical formation assay, 6-well plates were firstly covered using 2 mL bottom agar mixture with DMEM, 10 % fetal calf serum, and 0.6 % agar. After solidification of the bottom layer, top agar-medium mixture with 2 × 104 cells from each experimental group was added and incubated at 37 °C for 2 weeks, followed by crystal violet staining. The sphere (diameter ≥ 100 μm) numbers were calculated from 5 fields per well.
Cellular migration assay
For transwell study, each group of cell samples were firstly placed with density of 6 × 104 in the upper chamber (8-µm) (Corning, Lowell, MA, USA). And the bottom chamber was added with complete medium. Then after incubation for 48 h under 37 °C, 5 % CO2 condition, cells in the lower chamber were fixed and stained in crystal violet solution for observation using microscope (Olympus). For the number of penetrating cells calculation, ten randomly chose fields per sample were selected.
For wound healing assay, each group of cells were respectively seeded into 6-well plate. Cellular monolayers were scraped with a sterile micropipette tip. The wounded monolayers were then washed with phosphate buffer solution (PBS) to remove debris. The distance between the two edges of the wound was calculated at three different positions. The distance between the two edges should be measured again after 48 h of incubation.
Cellular survivability assay
Each of the cell group was respectively challenged with different concentrations of fluorouracil, cisplatin, doxorubicin for 4 h, and then cells were further washed by PBS and treated by 75 % ethanol at − 20 °C overnight. Then at room temperature, these cells were treated with Annexin V-FITC (Thermo Fisher Scientific) and propidium iodide (PI) for 20 min. Afterwards, flow cytometric analysis was performed (FACScan™, BD Biosciences, Franklin Lakes, NJ, USA) to detect apoptotic cells.
Statistical analysis
Statistical analysis was conducted by software package (SPSS 21.0 for Windows; IBM-SPSS Inc., Chicago, IL). Data were presented as the mean SD of three independent experiments. Statistical test of differences between numerical data was performed by standard t-test. Pearson test was conducted to compare gene correlation. p < 0.05 was considered to indicate statistical significance.
