Patient tissue samples and cell lines
A total of 50 paired pancreatic cancer tissues and adjacent normal tissue samples were obtained from patients that experienced surgery at the First Affiliated Hospital of Jinzhou Medical University from January 2012 to December 2015. Tissues were stored at liquid nitrogen before use. The research was approved by the Ethics Committee of The First Affiliated Hospital of Jinzhou Medical University. Informed consents were obtained from all the patients.
The human PCa lines AsPC-1, PANC-1, Sw1990 and BXPC-3 were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were grown in DMEM medium supplemented with 10% fetal bovine serum (FBS, Gibco, NY, USA), 100 U/mL penicillin and 100 μg/mL streptomycin (Gibco, NY, USA). Cells were maintained at 37ºC in a humidified atmosphere with 5% CO2.
RNA preparation and RT-qPCR
Total RNA was extracted from tissues or cell lines using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The synthesis of cDNA was performed by reverse transcription using the TaqMan MicroRNA Reverse Transcription Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. RT-qPCR was carried out using the TaqMan Universal PCR Master Mix Kit (Thermo Fisher Scientific, Waltham, MA, USA) with the ABI 7500 Fast Real-Time PCR System (Applied Biosystems). The thermocycling conditions were as follows: 95ºC for 5 min, followed by 40 cycles of 95ºC for 10 sec and 60ºC for 30 sec. The expression of U6 RNA was detected as the control. The primers were designed as: miR-26a forward, 5’-GACTGTTCAAGTAATCCAGGATA; miR-26a reverse, 5’-GTGCAGGGTCCGAGGTATTC; U6 RNA forward, 5’-CTCGCTTCGGCAGCACA; U6 RNA reverse, 5’-AAACGCTTCACGAATTTG CGT. The relative level of miR-26a was calculated using the 2-ΔΔCT method.
Cell counting kit-8 (CCK-8) assay for cell proliferation
The proliferation of PCa cells was assessed by the CCK-8 assay. Cells (1,000 cells/well) transfected with miR-26a mimic or miR-NC were seeded into the 96-well plates and cultured for 1-, 2-, 3-, 4- and 5 day. CCK-8 solution (Beyotime, Shanghai, China) was added into the medium at the indicated time points and incubated at 37ºC for 4 h. The absorbance at 450 nm was measured with the microplate reader (Bio-Rad Laboratories, Inc., USA).
Colony formation assay
PCa cells expressing miR-26a mimics or miR-NC were seeded into the 6-well plates with 500 cells per well. Cells were grown in DMEM containing FBS and maintained for 10 days. Cells were stained with 0.1% crystal violet (Beyotime, Shanghai, China) after fixation with methanol at room temperature (RT) for 10 min. The colonies were counted with the light microscopy.
Protein samples were prepared using the RIPA lysis buffer containing protease inhibitor (Beyotime, Shanghai, China). Equal amount of proteins were loaded and separated by 15% SDS-PAGE, and then transferred onto the PVDF membrane. After blocking with 5% non-fat milk, the membrane was incubated with the primary antibodies against E2F7 (1:1000 dilution; cat. no ab56022, Abcam) or GAPDH (1:3000 dilution; cat.no ab181602, Abcam) overnight at 4ºC. The membrane was then incubated with fluorescently labeled secondary antibodies and the bands were visualized using the Odyssey infrared scanner (Li-Cor Bioscience, Lincoln, NE, USA).
Dual-luciferase reporter assay
The wild-type (WT) or mutant (MT) 3’-UTR sequence of E2F7 was constructed into the pGL3 luciferase vectors (Promega, Madison, USA) and transfected into pancreatic cancer cells with miR-26a mimic or miR-NC. To detect the binding between E2F7 and VEGFA, the promoter sequence of VEGFA was inserted into the backbone of pGL-Basic (Promega, Madison, USA) vector. Cells were co-transfected with pGL-Basic-VEGFA and pcDNA-E2F7 or pcDNA-empty vector. After transfection for 48 h, cells were harvested and the luciferase activity was determined with the Dual-luciferase reporter assay kit (Promega, Madison, USA). The activity of renilla luciferase was also determined as the normalization. The experiment was performed in triplicates.
Chromatin Immunoprecipitation (ChIP) assay
The CHIP assay was performed with the protocol as previously described. Briefly, cells transfected with the indicated expression vectors were harvested after cultured for 48 h. Cells were cross-linked with 1% formaldehyde at RT for 10 min and then resuspended in lysis buffer containing protease inhibitor on ice for 10 min. Samples were then sonicated at 4ºC at the condition of 20 kHz to generate chromatin fragments of 200-500 bp. DNA fragments were purified using the QIAquick PCR purification kit (Qiagen, USA). Recovered DNA was used for qPCR amplification using the SYBR master mix (Bio-Rad, USA) with the primers of VEGFA (forward, 5′-GCTGTTTGGGAGGTCAGAAATAGG and reverse, 5′-ACGCTGCTCGCTCCATTCAC). Antibody against E2F7 (sc-H300, Santa Curz Biotechnology, USA) and normal rabbit IgG was used as a negative control.
Data was presented as the mean ± standard deviation. Data was analyzed with the SPSS 19.0 (IBM, Armonk, NY, USA). Difference was determined with the Student’s t test or One-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test. P<0.05 was considered as significant.