The pancreatic cancer cell lines PANC-1 and AsPC-1 were purchased from ATCC (USA). PANC-1 and AsPC-1 cell lines were cultured in DMEM (Thermo Fisher Scientific, USA) supplemented with 10% FBS (HyClone, USA) at 37°C in a 5% CO₂ incubator.
Antibodies, plasmids and chemicals
Human expression vectors for flag-tagged recombinant proteins were generated using the pcDNA3.1 backbone vector. The ITGA2 antibody (#ab133557, Abcam, USA); GAPDH (#10494–1-AP, Proteintech, USA). TFCP2(#15203-1-AP, Proteintech, USA); SMAD2 (#13684S, Cell Signaling Technology, USA). The TGF-β recombinant protein (#ab50036; Abcam, USA); KRAS(G12D) inhibitor (#S8499, SELLECK, USA),U0126 (#HY-12031A; MedChemExpress, USA).
Lipofectamine 3000 (Invitrogen, USA) and Opti-MEM media (Invitrogen, USA) were used for the transfection reactions; lipofectamine 3000 was used to transfect 293 T cells to shRNA plasmids and viral packaging plasmids (pVSV-G and pEXQV). 24h after transfection, the medium was replaced with fresh DMEM containing 10% FBS and 1mM sodium pyruvate and 48h post-transfection, the virus culture medium was collected and added to the PANC-1 and AsPC-1 cells supplemented with 12µg/ml of polybrene. 24h after infection, the infected cells were selected with 10µg/ml of puromycin. The sh-RNAs sequence information is provided in the Supplementary table 1.
Immunoprecipitation and Western blot analysis
The radioimmunoprecipitation assay (RIPA) lysis containing 1% protease inhibitor and phosphatase inhibitor was used to cleave the cells on ice, and the cell lytic products were obtained. After centrifugation with 12000rpm at 4 ℃ for 15 minutes, the undissolved impurities were removed and the supernatant was collected. The protein concentration was measured by BCA's experimental method. The protein extract and agarose beads with antibodies were shaken slowly at 4 ℃ and incubated overnight for co-immunoprecipitation test or Western blotting analysis. The precipitated immune complex was electrophoretic by SDS-PAGE, then transferred to a 0.45-µm polyvinylidene fluoride (PVDF) membrane, then blocked with 0.5% bovine serum albumin (BSA) and incubated with a specific first antibody, and then the membrane was then visualized by the electrogenerated chemiluminescence (ECL) method. Bio-rad microscopic imaging system was employed to capture images that were analyzed using Image Lab.
Reverse transcription quantitative polymerase chain reaction (RT-qPCR)
After total RNA was extracted from cells by Trizol reagent (Invitro-gen, 15,596,026, USA), the concentration of RNA was determined by spectrophotometer. RNA samples (1 µg) were reverse-transcribed using a PrimeScript™ RT reagent Kit (TAKARA, RR047A, JPN). Quantitative real-time PCR was performed using a TB Green™ Fast qPCR Mix kit (TAKARA, RR430A, JPN). Values represent the averages of three technical replicates from at least five independent experiments (biological replicates).
The primer sequences information for RT-qPCR is provided in the Supplementary table 2.
Liquid chromatography-tandem mass spectrometry/mass spectrometry (LC-MS/MS) analysis
293T cells transfected with a Flag-PTEN-expressing plasmid were used to identify novel PTEN-binding proteins. PTEN was immunoprecipitated using an anti-PTEN antibody and protein A+G agarose (#P2012, Beyotime, China) at 4°C. LC-MS/MS analysis was performed using a Thermo Ultimate 3000 liquid phase combined with Q Exactive Plus high-resolution mass spectrometry at Shanghai Applied Protein Technology. The data were retrieved using maxquant (v1.6.6) software and the algorithm Andromeda. The reference database comprised the UniProt human proteome reference database. Proteins and peptides with a false discovery rate (FDR) of 1% were selected.
In total, 1 µg of RNA per sample was used as the input material for RNA sequencing (RNA-seq). Sequencing libraries were generated using the NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, USA), in accordance with the manufacturer’s instructions. Clustering of the samples was performed on the cBot Cluster Generation System using the TruSeq PE Cluster Kit v3-cBot-HS (Illumina), in accordance with the manufacturer’s instructions. After cluster generation, libraries were sequenced on an Illumina Novaseq platform, and 150-bp paired-end reads were generated. FeatureCounts v1.5.0-p3 was used to count 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). The clusterProfiler R package was used to test the statistical enrichment of differentially expressed genes (DEGs) in KEGG pathways.
Colony formation assay
Colony formation assay can detect the biological effects of ITGA2 and TGF- β on tumor cell survival. Four stable PANC-1 and AsPC-1 cell lines of sh-Control, sh-ITGA2 and TGF- β-treated sh-Control and TGF- β-treated sh-ITGA2 were inoculated into six-well plates with 500 cells in each well, and repetitive group was set up. After 10-14 days of culture, the colony formation assay was fixed, stained and photographed.
CCK8 cell proliferation assay
Four stable cell lines were used to establish four treatment groups including control groups and experimental groups, and five repetitive groups were set up in each group. 2000 cells were added in each well and 10 microliters of CCK8 solution were added in each well. The cells were incubated in the incubator for 3 hours, and the absorbance was measured by enzyme labeling instrument in 450nm. The data were monitored continuously for five days, and finally the data were processed with GraphPad Prism 7.
Chromatin Immunoprecipitation (ChIP) Assay
The binding sites of ITGA2 and SMAD2 with TFCP2 were verified by CHIP experiment. Formaldehyde was added to the cells to make the target protein cross-linked with the genomic DNA, and the lysate digested the cells. The above samples were treated with ultrasound to break the genomic DNA to 200-100bp. After treatment, the target protein and its bound DNA fragment were pulled down by co-immunoprecipitation, and then purified and amplified by PCR. The primer sequences information for ChIP-qPCR is provided in the Supplementary table 3.
Two stable transfer cell lines (sh-control and sh-ITGA2) were collected in the centrifuge tube, and the cytoplasmic protein extraction reagent A with pre-added PMSF was added to the centrifuge tube. After shaking and mixing, the above samples were added to the cytoplasmic protein extraction reagent B in the ice bath 10min. Shake and mix well and take an ice bath for 1 minute. Centrifuge 12000g at 4 ℃ for 5 minutes. The supernatant was obtained in a precooled centrifuge tube, and the cytoplasmic protein was obtained. Nuclear protein extraction reagent with PMSF was added to the precipitation of the supernatant, and then 2min was mixed in an ice bath, followed by shaking for 20 seconds every 2 minutes for a total of 30 minutes. The supernatant of the above samples was centrifuged at 4 ℃ for 10 minutes, and the supernatant was obtained as nuclear protein. The concentrations of plasma protein and nuclear protein were measured by BCA method. Finally, the Western blotting analysis was carried out.
Four stable pancreatic cancer cell lines in logarithmic growth phase were digested with trypsin and collected in a flow tube with pre-added culture medium. After centrifugation 300g/5min, the supernatant was discarded. After PBS washing, the supernatant was re-suspended with 300 µl of Binding Buffer, 5 µl of Annexin V-FITC was added and incubated in the dark for 10 minutes, then 5 µl of PI was added, mixed and incubated in the absence of light for 5 minutes, and the corresponding channels were detected and observed within 1 hour.
The correlation between TFCP2 and ITGA2 and SMAD2 was analyzed by GEPIA (http://gepia.cancer-pku.cn/) database, and the binding sites of transcription factors TFCP2 of ITGA2 and SMAD2 were predicted by EPD (https://epd.epfl.ch//index.php). Path enrichment analysis of sequencing results using R language and GSEA analysis. The expression of ITGA2 in KRAS wild type and mutant pancreatic cancer was obtained by cBioPortal database (https://www.cbioportal.org/) query.
Mouse tumor model.
Animal experimental procedures were in accordance with guidelines of the Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology. 5 × 106 PANC-1 cells infected with sh-Control or sh-ITGA2 were injected subcutaneously into the left side of purchased BALB/c-nu mice (4-5 weeks old, male). The mice were intra-tumoral injected with TGF-β recombinant protein for 3 times on days 1, 4, and 7 at a dose of 10 mg/kg . The tumor size was evaluated with digital Vernier caliper every two days, and then tumor volumes were calculated using the formula: (L x W 2) / 2. Mice were sacrificed on day 21 or when tumor volume reached 1000 mm3.
All experiments were performed at least three times. Parametric data are shown as means ± standard errors of the mean (SEMs) and nonparametric data as medians and ranges. Two-way ANOVA or one-way ANOVA with Tukey’s multiple comparison test was used for multiple group analysis. Unpaired Student’s t-tests were used to compare data between two groups. Two-tailed P-values < 0.05 were considered statistically significant. All statistical analyses were performed using GraphPad Prism 6 software (GraphPad Software, Inc, USA).