Patient samples and clinicopathologic data
A total of 50 pairs of pancreatic cancer and adjacent non-tumor tissues were obtained from the Department of General Surgery in Xinhua Hospital affiliated with Shanghai Jiao Tong University (Shanghai, China) between 2012 and 2016. All cases hadn’t received any radiotherapy or chemotherapy before surgery, and were histologically confirmed and staged based on the American Joint Committee on Cancer Staging Manual (8th edition). The informed consent was obtained from all enrolled patients and the study was approved by Ethics Committee of Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine (Approval No. XHEC-D-2020-076).
Immunohistochemistry (IHC) staining was conducted following the standard staining procedure (16)(X et al., 2019). The ERRα expression level was scored based on the percentage of immunoreactive cells: Negative, < 5% immunoreactive cells; Weak, 5%-34% immunoreactive cells; Moderate, 35%-64% immunoreactive cells; Strong, ≥ 65% immunoreactive cells. The total staining score was based on the sum of the extent and intensity, and samples were classified as negative (0–1), weak (2–3), moderate (4–5) and strong (6–7) staining. Among all the PC tissues, 34 were identified as ERRα-high tissues (moderate and strong staining) and 16 were identified as ERRα-low tissues (negative and weak staining) according to the IHC score.
Cell culture and reagents
Pancreatic cancer cell lines (Mia PaCa-2, PaTu8988, PANC1), normal human pancreatic ductal epithelial cell line (HPNE) and 293T cell line were obtained from the Shanghai Key Laboratory of Biliary Tract Disease Research, Shanghai, China. All cell lines were cultured in high-glucose DMEM (Gibco) supplemented with 10% fetal bovine serum (Gibco), and incubated in a humidified incubator with 5% CO2 at 37℃.
GDC-0994 (HY-15947) was purchased from MedChemExpress and dissolved in dimethylsulfoxide (DMSO). The final DMSO concentration in culture medium was less than 0.1%. Mia PaCa-2 cells were pretreated with 5 µM or 10 µM GDC-0994 for 24 hours before further assays.
Quantitative real-time PCR (qRT-PCR)
Total RNA was extracted from patient samples and cells using Trizol reagent (Invitrogen). cDNA was synthesized using PrimeScript RT reagent kit with gDNA Eraser (Takara), and the levels of transcripts were detected using SYBR-Green method (Takara) by the StepOnePlus Real-Time thermocycler (Applied Biosystems) following the manufacturer’s instructions. The primers sequences are as follows: ERRα forward, 5′-CACTATGGTGTGGCATCCTG-3′ and ERRα reverse, 5′-CGCTTGGTGATCTCACACTC-3′; PAI1 forward, 5′-ACCGCAACGTGGTTTTCTCA-3′ and PAI1 reverse, 5′-TTGAATCCCATAGCTGCTTGAAT-3′; GAPDH forward, 5′-GGAGCGAGATCCCTCCAAAAT-3′ and GAPDH reverse, 5′-GGCTGTTGTCATACTTCTCATGG-3′.
ERRα was silenced with small interfering RNAs (siRNAs) using Rfect reagent (BAIDAI) according to the manufacturer’s protocol. The sense sequence are as follows: si-ERRα-1 (sense, GCGAGAGGAGUAUGUUCUA; antisense, UAGAACAUACUCCUCUCGC); si-ERRα-2 (sense, GAGAGGAGUAUGUUCUACUAA; antisense, UUAGUAGAACAUACUCCUCUC); si-PAI1 (sense, UCUCUGCCCUCACCAACAUUC; antisense, GAAUGUUGGUGAGGGCAGAGA). The shRNA targeting ERRα was synthesized using the si-ERRα-1 sequence and inserted into PGMLV-SC5 vector, and the full-length sequence of ERRα was cloned into pCDNA3.1 vector (Genomeditech, Shanghai). Empty vectors were used as control. Cells were infected by concentrated lentivirus at a multiplicity of infection (MOI) of 90 for 48 hours. Cell lines were selected by puromycin (1 µg/ml) for 1 week to construct stable transfected cell lines, transfection efficiency was verified by qRT-PCR and western blotting.
Cells were fixed with 4% paraformaldehyde, and immunofluorescence (IF) assay was conducted using Immunol Fluorence Staining Kit with Cy3 (Beyotime, China) following the manufacturer’s instructions. Briefly, cells were permeabilized in 0.1% Triton-X-100, blocked by 3% BSA, incubated with primary antibody at 4℃ overnight and then incubated with Labeled Goat Anti-Rabbit IgG at room temperature away from light. DAPI was used for cell counterstaining. A fluorescence microscope (Leica) was used to capture photos.
Western blot analysis
Protein extraction and western blot was performed according to the standard protocol as previously described (17)(S et al., 2019). In brief, equal amounts of proteins were separated by SDS-PAGE and transferred onto PVDF membranes. The membranes were blocked with 5% skim milk, incubated with specific primary antibodies at 4℃ overnight and then with HRP-conjugated secondary antibody at room temperature for 1 h. Protein signals were detected using a Gel Doc 2000 (Bio-Rad, USA). All primary antibodies were purchased from Cell Signaling Technology.
Cell proliferation assays
Cell Counting Kit-8 (CCK-8) assay and 5-Ethynyl‐2′‐deoxyuridine (EdU)-488 proliferation assay were performed to evaluate cell proliferation state. For CCK-8 assay, cells were seeded into a 96-well plate at the density of 2000/well with 100 µL complete medium overnight. Each well was then replaced with 10 µL CCK-8 reagent with 90 µL medium, incubated in dark at 37 ℃ for 2 hours and then using SpectraMax 190 Microplate Reader to measure the optical density at 450 nm.
EdU Cell Proliferation Kit (Beyotime) was used to detect cell proliferation according to the manufacturer's instructions. Cells were incubated with 10 µM EdU in complete medium at 37 ℃ for 2 hours. Azide 488 and Hoechst reagent was used for detection of EdU-positive cells and cell counting, respectively. Images were taken under a fluorescence microscope (Leica).
Colony formation assay
Transfected cells were plated onto 6-well plates (1000 cells/well) and cultured for 7 days. Then cells were fixed with 4% paraformaldehyde for 20 minutes and stained with 0.1% crystal violet stain solution for 15 minutes. Images of the stained plates were photographed and the colonies were counted.
Nude mice xenograft models
Female BALB/c nude mice (4 weeks old, weighted 18–22 g) were purchased from Shanghai Laboratory Animal Center of the Chinese Academy of Sciences. The mice were randomly divided into 4 groups (ERRα, vector, shERRα, vector-shRNA) of 5 mice each and housed under appropriate condition. 2 × 106 PaTu8988 cells with stable transfection of LV-ERRα, LV-shERRα or their empty vectors were injected into the left axilla of each mouse. The volume of tumor was measured with calipers weekly using the following formula: 0.5 × width2 × length. 4 weeks after the cells injection, all mice were sacrificed by cervical dislocation, and the xenograft tumors were carefully dissected and weighed. This animal study was approved by the Ethics Committee of Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine.
Transwell migration and invasion assays were applied using chamber inserts (Corning) and BioCoat Matrigel Invasion chamber inserts (Corning), respectively. Treated cells were suspended in 200 µL serum-free medium and seeded in the upper chamber. For both assays, the lower chamber was added with 750 µL complete medium. After 24 hours incubation, cells in the lower surface were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet stain solution. 5 random fields of views were photographed under a microscope.
Wound healing assay
Cells were seeded in 6-well plates and cultured to more than 90% cell confluence. Wounds were scratched with same strength by use a 200-µL pipette tip. After washing with fresh medium and incubating for 24 hours, images were taken by a microscope at 5 random fields of views. Wound closure percentage was evaluated by comparing the changes before and after 24 hours.
Annexin V-FITC Apoptosis Detection Kit (Beyotime) was used to detect cell apoptotic ratio following the manufacturer’s instructions. Transfected cells were harvested and resuspended with binding buffer, and stained with 5 µL Annexin V-FITC and 10 µL propidium iodide (PI). After 20 minutes incubation at room temperature away from light, stained cells were analyzed by flow cytometry (BD Biosciences).
Cell cycle analysis
Cell Cycle Analysis Kit (Beyotime) was used for analysis of cell cycle distribution. Transfected cells were collected and fixed with ice-cold 70% ethanol at 4 ℃ overnight. The next day, cells were incubated with RNase A and PI for 30 minutes. DNA content was measured by flow cytometry (BD Biosciences) and analyzed by FlowJo software.
Total RNA was extracted from PaTu8988 and PANC1 cells after transfection with si-NC or si-ERRα-1, and then subjected to mRNA sequencing on the BGISEQ-500 platform (Beijing Genomics Institute, China). Of all the differentially expressed genes, 40 most up-regulated genes and 40 most down-regulated genes were selected and analyzed using the BGI online analysis system.
The promoter sequence of PAI1 was obtained from NCBI (https://www.ncbi.nlm.nih.gov/), the potential binding sites of ERRα in the promoter region of PAI1 were identified by JASPAR (http://jaspar.genereg.net/). Chromatin immunoprecipitation (ChIP) was performed using the PaTu8988 cells and a ChIP Assay Kit (Beyotime) according to the manufacturer’s protocol. Briefly, cross-linked chromatin was sonicated into 200- to 1000-bp fragments, and then immunoprecipitated with normal rabbit IgG antibody (Cell Signaling Technology) or anti-ERRα antibody (Cell Signaling Technology). Precipitated ChIP samples were analyzed by 2% agarose gel DNA electrophoresis and quantitative PCR (qPCR). Primers for the 2 binding sites are as follows: site 1 forward, 5’-CTCCAACCTCAGCCAGACAA-3’ and site 1 reverse, 5’-CCTCCGATGATACACGGCTG-3’; site 2 forward, 5’-CTCCACAGTGACCTGGTTCG-3’ and site 2 reverse, 5’-CGGGTGACCCAAAAAGCCTA-3’.
Dual-luciferase reporter gene assay
293T cells transfected with full-length ERRα, shERRα or their empty vectors were seeded onto 24-well plates and co-transfected with indicated luciferase reporters. pGL3-PAI1-WT-luc (containing the − 2000 bp to -1 bp promoter sequence of PAI1) and pGL3-PAI1-MUT-luc (the 2 binding sites of ERRα in the promoter region of PAI1 were mutated) were synthesized by Genomeditech. At 48 hours post-transfection, luciferase activities were determined by a Dual-Luciferase Reporter Assay System (Promega) following the manufacturer’s instructions. The relative luciferase activities were presented after normalization to Renilla luciferase activity.
All experiments were repeated at least three times. Data were analyzed using Prism 8 software and presented as the mean ± standard deviations (SD). The Student’s test was performed for the comparisons between two groups, and Pearson chi-square test was used to analyze the correlation between ERRα expression and clinicopathologic variables. Survival analysis was applied by Kaplan-Meier method and log-rank test. P < 0.05 was considered statistically significant.