ITGBL1 expression in multiple databases
To evaluate the expression level of ITGBL1 in somatic tumors, Oncomine Timer, and UALCAN public databases were used first. The Oncomine database is an online website containing Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) integration for endogenous mRNA levels. The Timer server is an integrated resource mainly used to analyze immune-infiltrating cells, but it also allows researchers to estimate the differential expression for genes of interest between tumor and adjacent normal tissues across diverse cancer types. The UALCAN database was used to compare the expression level of ITGBL1 not only in stomach adenocarcinoma and normal tissues but also in different clinical classification subgroups including cancer stages, sex, age, tumor grade, HP infection status, and nodal metastasis status. The distributions of gene expression levels are displayed using box plots.
Survival analysis
Kaplan–Meier Plotter is a website for online survival analysis involving a variety of tumors for GEO, European Genome-phenome Archive, and TCGA databases, which was used to evaluate the OS rate of gastric cancer tissues based on high-throughput sequencing data or gene chip.
Prediction of association between TGBL1 and tumor-infiltrating immune cells
The relationship between six immune infiltrates (B cells, CD4+ T cells, CD8+ T cells, neutrophils, macrophages, and dendritic cells) and ITGBL1 was estimated using the Timer algorithm. Besides, another immune-related web server, TISIDB, was used to analyze the association between the abundance of tumor-infiltrating lymphocytes (TILs) and expression of ITGBL1 by comparing the Spearman correlation between ITGBL1 expression and TILs in human cancers.
Prediction of correlation between ITGBL1 and EMT-related factors
Timer and Starbase web servers were used to assess the correlation between EMT-related factors and ITGBL1 expression level in gastric cancer tissues. The two databases contained the routine data analysis of the mRNA of any gene of interest in the TCGA, including differential expression, survival analysis, and co-expression. The degree of correlation was computed using purity-adjusted partial Spearman's rho value. The correlations of ITGBL1 with these factors was finally shown with scatter plots.
Tissue specimens
The study included 43 matched tumor samples from patients with surgically resected GC from the First Affiliated Hospital of Bengbu Medical College in 2017 for clinical indications. This study was authorized by the research medical ethics committee of the First Affiliated Hospital, Bengbu Medical College and Tongji Hospital, Tongji University. Each patient signed an informed consent form. All tissue specimens were pathologically confirmed as GC.
Reverse transcription and quantitative real-time polymerase chain reaction
Total RNA was extracted with TRIzol reagent (Life Technologies, CA, USA). Subsequently, cDNA was synthesized by reverse transcription with PrimeScript RT kits (Takara Biotechnology Co., Japan) following the manufacturer’s protocol. Subsequently, generated cDNA was amplified with an SYBR PrimeScript RT-PCR kit (Takara Biotechnology Co.) on an ABI 7900 Real-Time PCR system. The primers used in the study were as follows: ITGBL1, (sense) 5'-AGACCTACGACGGGAGCAC-3' and (antisense) 5'-ACCTGCATTAGAGCAGATGATGT-3'; GAPDH, (sense) 5'-ATCACCATTGGCAATGAG-3' and (antisense) 5'-AAGGTAGTTTCGTGGATG-3'. The 2-comparative Ct (2-ΔΔCt) formula was used to calculate the relative expression levels of ITGBL1, and GAPDH was normalized as an internal control.
Immunohistochemical staining
Tissues from patients with GC were fixed with 10% formalin for 48 h and then embedded with paraffin. Followed by deparaffinization and rehydration, the tissues were treated with 3% H2O2 for 10 min prior to blocking in goat serum for 1 h. Subsequently, a specific primary antibody ITGBL1 (1:100, Proteintech, China) was used to incubate the sections at 4°C overnight. Next, horseradish peroxidase–labeled secondary antibodies were used to incubate tissue sections for 1 h at 37°C and analyzed by the SABC method. The evaluation of color development results was performed as previously described [40].
Cell culture
Human normal gastric epithelial cell line GES-1 and GC cell lines SGC7901, BGC823, and MGC803 were provided by the Cell Division Center in Tongji Hospital of Tongji University, Shanghai, China. They were cultured in 90% DMEM (Gibco) with 10% fetal bovine serum (FBS, Gibco) at 37°C and incubated in a humidified 5% CO2 atmosphere.
Oligonucleotide transfection
Further, 2.0 × 105 cells/well of MGC803 cell lines were seeded in a six-well plate prior to transfection. After reaching 50% fusion density, the cells were transfected with an ultimate concentration of 50nM of either negative control (NC) or ITGBL1-sh (targeted to interfere with ITGBL1) using Lipofectamine 2000 Reagent (Thermo Fisher Scientific, MA, USA). Three different oligonucleotide chains specifically targeting ITGBL1 were designed by Asia-Vector Biotechnology (Shanghai, China). The sequences were as follows: (sense) ITGBL1-sh1:5′-TGGGAAGTGTTACTGTGGA-3′, (antisense) 5′-TCCACAGTAACACTTCCCA-3′, (sense) ITGBL1-sh2: 5′- ACGATGAAACAGAAGAAAT-3′, (antisense) 5′- ATTTCTTCTGTTTCATCGT-3′, ITGBL1-sh3: (sense) 5′- GTGGACTTGTGTATGGTAA-3′, (antisense) 5′- TTACCATACACAAGTCCAC-3′.
Cell apoptosis assay
Transfected cells were reseeded at a density of 5 × 105 cells/well onto six-well plates and collected with 0.25% trypsin without EDTA. Next, the collected cells were washed twice with cold PBS prior to resuspending with 1 × binding buffer. After that, 5 μL of Annexin V–PE and 10 μL of AAD (Yeasen, China) were added to each cell suspension to stain cells and incubated for 30 min. After adding 385 µL of 1 × Annexin-binding buffer, the cell apoptosis was assessed by flow cytometry.
Transwell migration/invasion assay
For assessing migration, the cells were cultured in the upper compartment of a Transwell chamber of an 8-μm pore size insert (Corning, NY, USA) at a density of 1.5 × 104 cells/well in serum-free culture medium. For evaluating invasion, the cells were incubated in the upper chamber of the insert coated with Matrigel (Corning, NY, USA). The lower chambers of both experiments were treated with DMEM containing 20% FBS. Following 48 h incubation, the cells remaining on the upper compartment were wiped off with cotton swabs. The cells that migrated and invaded into the bottom chamber were stained with crystal violet for 30 min. The results of the quantitative analysis were assessed using Image J software.
Western blot analysis
BCA protein assay was performed to determine protein concentrations. Equal amounts of protein lysates were segregated with 10% polyacrylamide gels (Bio-Rad, CA. USA) and transferred to 0.45 µm nitrocellulose membranes (Bio-Rad). Then, 5% BSA was used to block membranes for 1 h prior to incubation with primary antibodies: ITGBL1 (1:1000, Proteintech, China), Vimentin (1:1000, Cell Signaling, MA, USA), E-cadherin (1:1000, Proteintech, China), Snail (1:1000, Cell Signaling), TGF-β1 (1:2000, Abcam, China), and GAPDH (1:2000, Proteintech, China) at 4°C overnight. Next, TBS containing 0.5% (w/v) Tween 20 buffer was used to wash membranes three times, and the membranes were incubated with the sheep anti-rabbit or sheep anti-mouse IgG secondary antibodies (1:10,000, LK, China) for 1 h. Subsequently, the protein bands were detected using an ECL kit (Thermo Scientific) following the manufacturer’s protocol. The GAPDH antibody served as a control.
Statistical analysis
Each experiment was performed at least in triplicate. Data were expressed as means ± SEM. The discrepancies of grouping variables were analyzed using the Student t test. All statistical analyses were performed using SPSS 20.0, and P <0.05 indicated a statistically significant difference.