The mRNA expression data and overall survival data of AFAP1, AFAP1L1 and AFAP1L2 in The Cancer Genome Atlas (TCGA) STAD database were downloaded from the UCSC Xena browser (http://xena.ucsc.edu/) . GSE27342  in the Gene Expression Omnibus (GEO) database was used to analyze mRNA expression of AFAP family members. We also analyzed the effect of AFAP family members on survival of GC patients in Kaplan-Meier Plotter website (http://kmplot.com/analysis/) . The association between AFAP family members and clinicopathological characteristics and uni-/multi-variate analyses used to identify independent risk factors were both analyzed in Xiantao academic online analysis tool (https://www.xiantao.love/) using TCGA STAD database (https://portal.gdc.cancer.gov/). Gene Set Enrichment Analysis (GSEA) method was adopted to conduct Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses using TCGA STAD database. GSEA was performed using the clusterProfiler R package with false discovery rate (FDR) < 0.25 and P < 0.05 as the cut-off criteria.
Patients and specimens
A total of 40 pairs of randomly selected snap-frozen GC tissues and adjacent nontumorous gastric tissues (ANGTs) from initial surgical resection were obtained from Hunan Provincial People’s Hospital/The First Affiliated Hospital of Hunan Normal University and were used to qRT-PCR and western blot analysis. Meanwhile, these fresh GC tissues were fixed with formalin and paraffin embedded and further used to immunohistochemistry analysis. All patients were diagnosed with GC by two independent pathologists.
RNA extraction and qRT-PCR
TRIzol™ Reagent (ThermoFisher Scientific, Waltham, MA) was used to isolate total RNA according to manufacturer’s protocol. cDNA was synthesized using the ReverTra Ace® qPCR RT Master Mix (Toyobo, Japan) according to manufacturer’s instructions. qRT-PCR was performed using the SYBR® Green Realtime PCR Master Mix-Plus kit (Toyobo, Japan) in QuantStudio3 System (Applied Biosystems, CA). GAPDH was used as internal control. The mRNA expression level was calculated by 2−ΔCt or 2−ΔΔCT method based on cycle threshold (Ct). The experiments were done in triplicates. The primer sequences used in this study were listed in Additional file 1: Table S1.
Protein extraction and western blot
Total protein was extracted by RIPA lysis buffer (NCM, Soochow, China) containing Protease and Phosphatase Inhibitor Cocktail (NCM, Soochow, China). Equal amounts of total protein sample were separated by SDS-PAGE gel (NCM, Soochow, China) and further transferred to PVDF membrane (Millipore, Bedford, MA). The membranes were incubated with appropriate primary antibodies and then HRP conjugated secondary antibodies. β-actin was used as loading control. All the primary antibodies used in this study were listed in Additional file 1: Table S2.
IHC assay was conducted using streptavidin-peroxidase method (ZSGB-BIO, Beijing, China). In brief, clinical sample sections (4 μm) were firstly dewaxed, hydrated and antigen retrieved. After inactivating endogenous peroxidase, the sections were blocked with normal goat serum and incubated with appropriate primary antibodies overnight at 4 °C. Then, the sections were sequentially incubated with reaction enhancement solution, secondary antibodies solution and DAB peroxidase substrate. Finally, the sections were stained by hematoxylin, and further dehydrated and mounted. The primary antibodies used for IHC are listed in Additional file 1: Table S2. IHC score was calculated according to proportion and intensity of positive cells [20, 21], namely IHC score = intensity score × percentage score. The score of staining intensity: 0, negative; 1, weak; 2, moderate; 3, strong. The score of positive cells: 0, less than 5% positive cells; 1, 5 ~ 25% positive cells; 2, 26 ~ 50% positive cells; 3, 51 ~ 75% positive cells; 4, > 75% positive cells.
Cell lines and cell culture
Human gastric mucosal epithelial cell line GES-1 and five GC cell lines (AGS, MKN74, MGC-803, HGC27 and MKN45) were purchased from the Type Culture Collection of the Chinese Academy of Sciences (China) and maintained in RPMI-1640 medium (BioInd, Beit Haemek, Israel) supplemented with 10% FBS (BioInd, Beit Haemek, Israel) and 1% penicillin/streptomycin (BioInd, Beit Haemek, Israel) in humidified incubator at 37 °C with 5% CO2. Short tandem repeat (STR) analysis was used to authenticate all cell lines before experiments and all mycoplasma test were negative.
Vector construction and transfection
Lentiviruses containing AFAP1L1/VAV2/ITGA5 short hairpin RNAs (shRNA) or their open reading frame (ORF) and corresponding negative control lentiviruses were purchased from WZ Biosciences (Shandong, China). Transfection was performed according to manufacturer’s instructions. The transfection of lentiviruses was performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). Puromycin (2 µg/mL) was used to select stable clones. The knockdown and overexpression efficacy of corresponding lentiviruses were verified by qRT-PCR and western blot. The sequence of VAV2 shRNA was as follow: 5′-GCATGACTGAAGATGACAAGA-3′. The sequence of ITGA5 shRNA was as follow: 5′-GCAGAGAGATGAAGATCTACC-3′. The sequences of AFAP1L1 shRNA were as follows: shRNA1: 5′-GGGCGCAACTCCTTCCTATAT-3′; shRNA2: 5′-GGTGTGGGTGACAACTGTTCT-3′; shRNA3: 5′-GCAAGTCGCCTGAGTACATCA-3′.
In vitro cell proliferation assays
CCK8 assay, colony formation assay and Edu assay were performed to test the role of AFAP1L1 on GC cell proliferation. For CCK8 assay, around 2 × 103 cells were seeded into 96-well plates. After cell attachment, the cells were incubated with CCK8 solution for 2 h and subjected to determine absorbance at 450 nm using spectrophotometer. For colony formation assay, around 5 × 102 GC cells were added into 6-well plates and cultured in incubator for 2 weeks. Then, the colonies were stained by crystal violet (Beyotime Biotechnology, Shanghai, China) and counted for diameter more than 40 μm. The Edu assays were performed using Cell-Light EdU Apollo567 In Vitro Kit (RiboBio, Guangzhou, China) according to the manufacturer’s protocol. The percentage of Edu positive cells was calculated and compared. All experiments were repeated three times.
GC cells were seeded into 6-well plates inserted with glass coverslip. After attachment, cells in the coverslip were fixed by 4% formaldehyde and perforated by 0.5% Triton X-100. Then, the cells were incubated with primary antibody overnight at 4 °C. Appropriate fluorescence labeled secondary antibodies were used to detect corresponding proteins. In addition, rhodamine-conjugated phalloidin (Roche, Basel, Switzerland) was used to detect cytoskeleton of GC cells. Nuclei were stained by DAPI solution (Beyotime Biotechnology, Shanghai, China). The primary antibodies for immunofluorescence used in this study were listed in Additional file 1: Table S2.
Transwell migration and invasion assays
For transwell migration assay, the suspended GC cells were added into upper chamber of transwell insert (Corning, Kennebunk, ME) without Matrigel and cultured for 24 h. For transwell invasion assay, GC cells were added into upper chamber of the transwell insert (Corning, Kennebunk, ME) with Matrigel (BD Biosciences, Franklin Lakes, NJ) and cultured for 24 h. The cells that invaded to bottom membrane of chamber were fixed and stained with 0.1% crystal violet (Beyotime Biotechnology, Shanghai, China). Mitomycin-C (10 µg/mL, Sigma, St. Louis, MO) was used to suppress cell proliferation before the experiments. All experiments were repeated three times.
HCC mouse model and in vivo study
All in vivo studies were performed using 6-week-old male BALB/c nude mice. The subcutaneous mouse model was used to detect GC cell growth in vivo. In short, around 5 × 106 cells within 200 μL cool PBS were injected subcutaneously into left upper flank region of nude mice. The subcutaneous tumor volume was calculated after 6 weeks using Vernier caliper as follow: tumor volume (mm3) = (L × W2)/2, where L = long axis and W = short axis. For in vivo metastatic assay, nude mice were injected with 1 × 106 GC cells (resuspended in PBS) via tail vein. After four weeks, all the lungs and livers were fixed with formalin and sectioned serially and stained with hematoxylin and eosin (H&E) for histological examination. The number of metastatic nodules in mice lung and liver was analyzed and compared.
Cell adhesion assay
About 1 × 104 suspended GC cells were placed into each well of a collagen-coated 96-well plate and cultured at 37 °C for 1 h. Then, cells that did not adhere to the plate were washed off with PBS. The remaining GC cells bound to collagen on plate were stained by SRB Assay Kit (Abcam, Cambridge, England) according to the manufacturer’s protocol. The final absorbance was measured at 565 nm. Plates incubated with bovine serum albumin (BSA) were used as negative control. All experiments were repeated three times.
Co-IP was performed using a Pierce™ Classic Magnetic IP/Co-IP Kit (ThermoFisher Scientific, Waltham, MA) following manufacturer’s protocol. In brief, monolayer GC cells were lysed and centrifuged and the protein concentration was tested. Then, a total of 1000 μg proteins were incubated with 10 µg of IP antibody (AFAP1L1 or VAV2) or control IgG at 4 °C overnight to form the immune complex. After washing magnetic beads using IP Lysis/Wash Buffer, the protein sample/antibody immune complex was added into the washed magnetic beads and incubated at room temperature for 1 h with mixing. Finally, the target antigen was disassociated from the beads using Elution Buffer and the samples were analyzed by western blot.
The level of active CDC42 (GTP-CDC42) and active Rac1 (GTP-Rac1) in GC cells was measured using Rac1/Cdc42 Activation Assay Kit (Millipore, MA) according to the manufacturer’s protocol. Briefly, GC cells were lysed by lysis buffer. PAK1-PBD agarose beads, which specifically bound to active CDC42 and Rac1, were added into the cell lysates. Agarose beads were then washed with lysis buffer three times. Active CDC42 and Rac1 was disassociated from PAK1-PBD agarose beads by reduced sample buffer and boiled for 5 min. The final samples were analyzed by western blot using anti-CDC42 antibody or anti-Rac1 antibody. Similarly, the level of active RhoA (GTP-RhoA) in GC cells was measured by RhoA Pull-down Activation Assay Biochem Kit (Cytoskeleton, CO) using Rhotekin-RBD bead according to the manufacturer’s protocol.
Statistical analyses were performed using SPSS 20.0 (SPSS Inc., Chicago, IL) or Graphpad Prism 8 (GraphPad Software, San Diego, CA). The differences between two groups were analyzed by Student’s t test when the variance is homogeneous or Mann-Whitney U test when the variance is inhomogeneous. Categorical data was analyzed using Chi-square test or Fisher exact test. Correlation coefficient was determined by Pearson correlation analysis. Survival curves were depicted by Kaplan-Meier method and compared by log-rank test. Univariate and multivariate analyses were performed to determine prognostic factors based on Cox proportional hazards regression model. Data were expressed as mean ± SD from at least three independent experiments. P < 0.05 (two-tailed) was considered statistically significant.