EZR expression analysis
We searched the GEO and TCGA databases to download pancreatic cancer data and compare the human pancreatic cancer tumor samples with normal samples. GEO datasets (GSE10144845 and GSE107610) and TCGA dataset were utilized to measure the gene expression of EZR, then we obtained the heat map using RStudio software. GEPIA (http://gepia.cancer-pku.cn/) was used to analyze RNA sequencing expression data from samples (contains tumor and normal) of TCGA. The screening conditions contains: (1) selecting datasets: pancreatic cancer; (2) gene: EZR; (3) expression DIY:boxplot; (4) matched normal data. Survival analysis was also performed using online web server GEPIA (http://gepia.cancer-pku.cn/).
Construction of protein–protein interaction network
Protein-protein interaction (PPI) network of EZR was established through Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database. Correlation genes of EZR were screened using Gene Ontology (GO)-based functional analysis.P < 0.05 was set as the cut-off criterion.
Gene set enrichment analysis
the Gene Set Enrichment Analysis (GSEA) software (version 3.0) and Java software were used to analysis EZR. The “uniq.symbol.txt” data set was downloaded, the high-to-low grouped expression profile data was enriched and analyzed by default weighted enrichment statistics.
Patient data and tissue sample
The PC tissues and paired paracancer tissue were obtained from 26 patients who underwent surgical treatment between 2018 and 2020 at the Affiliated Hospital of North Sichuan Medical college, nanchong, China. All tissue were confirmed by histopathological examination and snap frozen in liquid nitrogen immediately after operation and then stored at -80°C. None of the patients have received any local or systemic therapy before surgery. All patients registered were informed and consent. All experiments were permitted by the research ethic committee of North Sichuan Medical college.
PC and paracancer tissues were collected and fixed in 10% formalin. After a 15-min antigen retrieval protocol in 0.01 M sodium citrate buffer (pH 6.0) at room temperature, the sections were blocked in 0.3% H2O2 and incubated for 1 h with Rb-EZR. Then, the sections were labeled with DAB and stained with hematoxylin. The sections were baked again prior to imaging,relevant analysis using a microscopic image analysis system (DS-Ri2, NIKON). The results were evaluated as 0, negative;1,weakly positive;2, moderately positive; or 3,strongly positive. we used the accurate formula to calculated the staining index:staining index=staining intensity ×percentage of positive cells. Low expression was defined as a staining index < 5.
PANC-1 and MIA PaCa-2 cell lines were purchased from ATCC (Manassas, USA), Human normal pancreatic ductal cells(HPDE), AsPC-1 and BxPC-3 cell lines were purchased from CASCB(Shanghai, China). All cells were grown in DMEM(Gibico, Carlsbad, CA, USA) medium supplemented with 10% fetal bovine serum (Gibico, Carlsbad, CA, USA) and 1% penicillin/streptomycin ((Beijing Solarbio Science, Beijing, China) at 5% CO2 and 37 °C.
EZR siRNAs and scrambled negative control (NC) siRNAs were purchased from RiboBio, Guangzhou, China. The cells were seeded and cultured in six-well plate with density of 3×105/well overnight. Then, cells were transfected with siRNAs or negative control at a final concentration of 50 nM using Lipofectamin 2000 reagent (Invitrogen, Carlsbad, U.S.A.) .
Quantitative real-time PCR (QRT-PCR) analysis
Total RNA was extracted from all cells, a TRIzol kit was used(Invitrogen, Carlsbad, CA, USA). Complementary DNA (cDNA) was used 2 μg of the total RNA according to the instructions of the reverse transcriptase kit (Takara Bio, Inc., Dalian, China) in a LifePro Thermal Cycler (Hangzhou Bioer Technology Co. Ltd., Hangzhou, China) . SYBR Premix Ex Taq (Takara, Japan) was used for Quantitative real-time PCR assay on the CFX Connect Real-Time System (Bio-Rad, U.S.A.). β-actin was used as the an internal reference gene for normalization. The forward and reverse primers for EZR were 5’-ACCAATCAATGTCCGAGTTACC-3’and 5’-GCCGATAGTCTTTACCACCTGA-3’, respectively. the primers for GAPDH were 5’-AACGGATTTGGTCGTATTGG-3’and 5’-TTGATTTTGGAGGGATCTCG-3’, respectively. Relative expression levels of genes were calculated by using the comparative cycle threshold (Ct) (2-ΔΔCt) method and then converted to fold-changes.
Cell proliferation and metastasis assay
A cell proliferation assay was implemented with a CCK-8 assay kit (Dojindo Laboratories Co. Ltd, Kumamoto, Japan) .Briefly, cells (5×103 cells/well) were seeded into 96-well plates with 100ul per well of DMEM culture medium supplemented with 10% FBS and cultured at 37°C and 5% CO2 atmosphere. Each sample has 6 replicates. The medium was replaced by 100 μl fresh culture medium, and 10 μl CCK-8 solution was added to each well for different periods of time (0, 24, 48 , 72 and 96 hours). Each well was measured spectrophotometrically at 450 nm using a Quant ELISA Reader (BioTek Instruments, USA) after 2 hours of incubation. All experiments were performed in quintuplicate and repeated once. Cell culture medium added to the bottom chamber by using 20% FBS,after 24 h,PC Cells were fixed ,stained with 4% paraformaldehyde and 0.4% crystal violet, respectively.50 μL Matrigel was pre-coated in the upper chamber for migration assay, and using the similar procedure to invasion assay.
Colony formation assay
For the colony formation assay, Cells (500 cells/well) were seeded into 6-well plates maintained in DMEM media containing 10% FBS for 2 weeks replacing the medium every 4 days. After fixation in 4% paraformaldehyde for 10 minutes, cells were stained with 1% crystal violet. Colonies with diameters greater than 100 μm were counted. Each sample was assessed in triplicate.
Co-Immunoprecipitation and Western blot analysis
Co-Immunoprecipitation(IP): PC cells were harvested，then resuspended in RIPA for 15 min. Cell lysate was centrifuged at 14000 r.p.m for 15 min. The supernatant incubated with primary antibody (or IgG) and Pierce Protein G Agarose,The beads were washed and resuspended with sample ,then heated at 100 °C for 5 min. quantified total protein with BCA method.Equal amount of lysate was separated and then transferred to the PVDF membrane. The membrane with target protein was incubated with the primary antibody at 4 °C and the second antibody was incubated for 1 h at room temperature. For Western blotting, total protein was extracted using RIPA with a Protease Inhibitor Cocktail. Then, the protein samples were transferred onto a PVDF membrane, which was followed by blocked with 5% fat-free milk at room temperature for 2 h, and an incubation overnight at 4 °C in a 1:500 dilution of primary antibodies. The membranes were washed three times with Tris-buffered saline containing Tween-20 (TBST), and then the membrane was incubated with HRP-conjugated rabbit or mouse secondary antibodies for 2h. The intensity of the protein band was densitometrically quantified using Image J software (version 1.50i).
All statistical analyses were performed with the SPSS 22.0 statistical software package (IBM, Chicago, USA) and GraphPad Prism 6.0 software(GraphPad software,USA). Two-sided P values were calculated, and a threshold of P<0.05 was considered statistically significant. The results are expressed as mean ± SD. Statistical significance was assigned at *P < 0.05 or **P <0.01. All experiments were carried out at least 3 times, in triplicate samples.