Cell culture
Pancreatic cancer cell lines PANC-1 and AsPC-1 were purchased from American type culture collection (ATCC, USA). The cell lines were cultured in Dulbecco’s modified eagle medium (DMEM, Thermo Fisher Scientific, USA) supplemented with 10% fetal bovine serum (FBS, HyClone, USA) and incubated at 37 °C with 5% CO2 concentration.
Antibodies, plasmids, and chemicals
Human expression vectors for the flag-tagged recombinant proteins were constructed using the pcDNA3.1 backbone vector. The antibodies for ITGA2 (#ab133557, Abcam, USA), GAPDH (#10494–1-AP, Proteintech, USA), TFCP2 (#15203-1-AP, Proteintech, USA), and SMAD2 (#13684S, Cell Signaling Technology, USA) and the recombinant proteins, including TGF-β (#ab50036; Abcam, USA), KRASG12D inhibitor (#S8499, SELLECK, USA), and U0126 (#HY-12031A; MedChemExpress, USA) recombinant proteins were purchased from the respective companies.
RNA interference
Lipofectamine 3000 (Invitrogen, USA) and Opti-MEM media (Invitrogen, USA) were used for the transfection studies. For this purpose, 293 T cells were transfected with shRNA plasmids and viral packaging plasmids (pVSV-G and pEXQV) using lipofectamine 3000. After 24 h of transfection, the medium was replaced with fresh DMEM, containing 10% FBS and 1 mM sodium pyruvate. After 48 h of transfection, the virus culture medium was collected and added to the PANC-1 and AsPC-1 cells, which were supplemented with 12 μg/ml of polybrene. After 24 h of infection with virus culture medium, the infected PANC-1 and AsPC-1 cells were selected using 10 μg/ml of puromycin. The sequences of shRNAs are provided in Supplementary Table 1.
Immunoprecipitation and Western blot analysis
Radioimmunoprecipitation assay (RIPA) lysis buffer, containing 1% protease inhibitor and phosphatase inhibitor was used for the cells’ lysis on ice and the cell lytic products were obtained. After centrifugation at 12000 rpm at 4 °C for 15 min, the undissolved impurities were removed and the supernatant was collected. The concentration of protein contents was measured using BCA’s experimental method. The protein extract was incubated overnight with agarose beads having antibodies in a slow-shaking incubator at 4 °C for co-immunoprecipitation test or Western blot analysis. The precipitated immune complexes were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a 0.45-μm polyvinylidene fluoride (PVDF) membrane. The membrane was then blocked with 0.5% bovine serum albumin (BSA) and incubated with the specific primary antibodies. The membrane was then visualized using the electrogenerated chemiluminescence (ECL) method. Bio-Rad microscopic imaging system was used to capture images, which were then analyzed using Image Lab software.
Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)
After total RNA extraction from the cells using TRIzol reagent (Invitrogen, 15,596,026, USA), the RNA concentration was determined using a spectrophotometer. The RNA samples (1 μg) were reverse-transcribed using a PrimeScript™ RT reagent Kit (TAKARA, RR047A, JPN). RT-qPCR was performed using a TB Green™ Fast qPCR Mix kit (TAKARA, RR430A, JPN). The data were presented as the average of three technical replicates from at least five independent experiments (biological replicates).
The primer sequences for the genes are provided in Supplementary Table 2.
Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis
The 293 T cells transfected with a Flag-PTEN-expressing plasmid were used for the identification of novel PTEN-binding proteins. The PTEN protein was immunoprecipitated using an anti-PTEN antibody and protein A + G agarose beads (#P2012, Beyotime, China) at 4 °C. LC-MS/MS analysis was performed using a Thermo Scientific Ultimate 3000 RSLC system combined with Q Exactive Plus high-resolution mass spectrometer by Shanghai Applied Protein Technology. The data were retrieved using MaxQuant (v1.6.6) software and the Andromeda algorithm. UniProt human proteome database was used as a reference database. The proteins and peptides with a false discovery rate (FDR) of 1% were selected.
RNA sequencing
A total of 1 μg extracted RNA sample was used for RNA sequencing (RNA-seq) per sample. The sequencing libraries were generated using the NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, USA), following the manufacturer’s instructions. The clustering of the samples was performed using the cBot Cluster Generation System with the TruSeq PE Cluster Kit v3-cBot-HS (Illumina), following the manufacturer’s instructions. After clustering, the libraries were sequenced on an Illumina NovaSeq platform, generating 150-bp paired-end reads. FeatureCounts v1.5.0-p3 software was used for counting the read numbers mapped to each gene. Differential expression analysis (two biological replicates per condition) was performed using the DESeq2 R package (1.16.1). ClusterProfiler R package was used to test the statistical enrichment of differentially expressed genes (DEGs) in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways.
Colony formation assay
Colony formation assay was used for detecting the biological effects of ITGA2 and TGF-β on the survival of tumor cells. Four stable PANC-1 and AsPC-1 cell lines groups, including sh-Control, sh-ITGA2, TGF- β-treated sh-Control, and TGF- β-treated sh-ITGA2, were inoculated into six-well plates with 500 cells per well; all the cell lines groups were set up in replicates. After 10-14 days of culture, the colony formation assay was fixed, stained, and photographed.
Cell counting Kit-8 (CCK8) cell proliferation assay
The four stable cell lines were used for four treatment groups, including control and experimental groups, and five replicates were set up for each group. A total of 2000 cells and 10 μL of CCK8 solution were added to each well. The cells were incubated in the incubator for 3 h and the absorbance at 450 nm was measured using an enzyme labeling instrument. The data were monitored continuously for 5 days and the final data were processed using GraphPad Prism v7.
Chromatin Immunoprecipitation (ChIP) assay
The binding sites of ITGA2 and SMAD2 with TFCP2 were determined using a CHIP assay. For this purpose, formaldehyde was added to the cells for cross-linking the target proteins with genomic DNA and digesting the cells to form lysate. These samples were then treated with ultrasound to break the genomic DNA into 200-1000 bp fragments. The target proteins and the DNA fragments bound to them were co-immunoprecipitated, purified, and amplified using PCR. The primer sequences for ChIP-qPCR are provided in Supplementary Table 3.
Nuclear-cytoplasmic separation
Two transfected cell lines (sh-control and sh-ITGA2) were collected in a centrifuge tube, to which, the cytoplasmic protein extraction reagent A with pre-added phenylmethanesulfonyl fluoride (PMSF) was added. After shaking and mixing, the cytoplasmic protein extraction reagent B was added to the samples and incubated on ice for 10 min. After shaking and mixing, the samples were centrifuged at 12000 g and 4 °C for 5 min. The supernatants were collected in the precooled centrifuge tubes and the cytoplasmic proteins were obtained, while the pellets were collected for nuclear proteins extraction. The nuclear protein extraction reagent with pre-added PMSF was added to the pellet. The samples were then incubated in the ice bath for 2, followed by shaking for 20 s; this process was repeated for a total of 30 min. The above samples were then centrifuged at 1000 g, 4 °C for 10 min, and the supernatants were obtained as nuclear proteins. The concentrations of cytoplasmic and nuclear proteins were measured using BCA. Finally, a Western blot analysis was carried out.
Flow cytometry
The four stable pancreatic cancer cell lines were digested with trypsin in their logarithmic growth phase and collected in a flow tube with a pre-added culture medium. After centrifugation at 300 g for 5 min, the supernatants were discarded. After washing with PBS, the pellets were re-suspended in 300 μL of binding buffer, to which, 5 μL of Annexin V (FITC) was added and incubated in dark for 10 min. Then, 5 μL of propidium iodide (PI) was added, mixed, and incubated in dark for 5 min. The corresponding channels were detected and observed within 1 h.
Bioinformatics mining
The correlations among TFCP2, ITGA2, and SMAD2 were analyzed using GEPIA (http://gepia.cancer-pku.cn/) database. The binding sites of TFCP2 with ITGA2 and SMAD2 were predicted using EPD (https://epd.epfl.ch//index.php). The pathway enrichment analysis of the RNA-Seq results was performed using R language and gene set enrichment analysis (GSEA) analysis. The expression patterns of ITGA2 in the KRAS wild-type and mutant pancreatic cancer cells were obtained using the cBioPortal database (https://www.cbioportal.org/) query.
Mouse tumor model
All the animal experiments were performed following the guidelines of the Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology. Approximately, 5 × 106 PANC-1 cells infected with either sh-Control or sh-ITGA2 were injected subcutaneously into the left side of BALB/c-nu mice (4-5 weeks old male mice). The intra-tumoral injection of TGF-β recombinant protein was performed 3 times on days 1, 4, and 7 at a dose of 10 mg/kg [11]. The tumor size was measured using a digital vernier caliper every 2 days and the tumor volumes were calculated using the formula (L x W2)/2. The mice were sacrificed on day 21 or when tumor volume reached 1000 mm3.
Immunofluorescence and immunohistochemistry staining
The cells were divided into two groups, including the treatment group (sh-ITGA2) and the control group (sh-control). The tissue sections were fixed with 4% PFA for 20 min at room temperature. After washing with PBS, the tissue sections were sequentially transferred to Triton-X-100 and 5% BSA solutions for 30 min, respectively. After washing three times with PBS for 5 min each, the tissue sections were incubated with rabbit anti-ITGA2 antibody and rabbit anti-TFCP2 antibody at a concentration of 1:1000 for 16 h. The sections were then rinsed with PBS three times and incubated with the respective fluorochrome-conjugated secondary antibodies for 1 h at room temperature. DAPI (4′,6-diamidino-2-phenylindole) counterstaining was performed to stain the nuclei. The primary antibodies used included anti-ITGA2 antibody (1:200 dilution; Abcam) and anti-TFCP2 antibody (1:200 dilution; Proteintech). For studying the immunohistochemistry, the tumor tissue sections were dehydrated with ethanol, washed with xylene, and mounted, followed by immunostaining of the sections in replicates. In order to quantify the status of Ki67 protein expression, each tissue section was visualized and the average percentage of positive cells was determined in at least 5 random fields. Fluorescence images were obtained using an immunofluorescent microscope (Olympus IX71, Japan) and a confocal laser-scanning microscope (LSM780, ZEISS, Germany).
Statistical analyses
All the experiments were performed in at least three replicates. Parametric data are shown as means ± standard errors of the mean (SEMs), while the nonparametric data are shown as medians and ranges. Two-way or one-way analysis of variance (ANOVA) with Tukey’s multiple comparison test was used for the multiple group analysis. Unpaired Student’s t-tests were used to compare the data between two groups. Two-tailed P-values < 0.05 were considered statistically significant. All the statistical analyses were performed using GraphPad Prism 6 software (GraphPad Software, Inc., USA).