TCGA analysis of ANP32E expression in pancreatic patients
The transcript level of ANP32E in pancreatic cancer tissues and normal tissues and the survival information of the patients were downloaded from The Cancer Genome Atlas (http://cancergenome.nih.gov) database. A total of 179 tumor tissues and 175 normal tissues were included to analyze the expression of ANP32E. A total of 89 patients with ANP32E high expression and 89 patients with low expression was included to determine the correlation between ANP32E expression and patients’ survival.
Human normal ductal epithelial cells of the pancreas HPDE and pancreatic cancer cells AsPC1, PANC1 and MIA were obtained from ATCC. The cells were cultured in Dulbecco modified Eagle’s medium (DMEM, Hyclone), which contained 10% fetal bovine serum (FBS, Gibco) and 1% penicillin and streptomycin (Corning). The cell culture was maintained in a 37oC incubator with 5% CO2.
Lentivirus vector system pGCSIL-GFP (with a GFP marker), Helper2.0 (VSVG element) and pHelper1.0 (gag/pol element) was used to knock down ANP32E in PANC1 and MIA cells. Targeting sequence of the Ctrl, ANP32E#1 and ANP32E#2 was 5’-TTCTCCGAACGTGTCACGT-3’, 5’-GTCCACCGGAAGGATATGA-3’ and 5’-GCCTCTCATACTTAATGAA-3’. The lentivirus was packaged by co-transfecting the pGCSIL-GFP, Helper2.0 and pHelper1.0 to 293FT cells. The knockdown efficiency was detected by qRT-PCR and Western blot.
Lentivirus vector system pCDH, PSPAX2 and PDM2G was used to over-express ANP32E in PANC1 and MIA cells. Coding sequence of ANP32E was cloned into the pCDH vector, which contained a GFP marker. Lentivirus was packaged by co-transfecting the pCDH, PSPAX2 and PDM2G to 293FT cells. Overexpression abundance was detected by qRT-PCR and Western blot.
The siRNAs against CTNNB or negative control were synthesized from GenePharma company. The transfection of the siRNAs were conducted using RNAimax (Invitrogen), following the following to the manufacturer’s instructions. The targeted sequence of siRNA was as followed: siCTNNB, 5’-CCCACTAATGTCCAGCGTT-3’; and siCtrl, 5’-TTCTCCGAACGTGTCACGT-3’.
RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR)
PANC1 and MIA cells were lysed using Trizol regent. Total RNA was extracted from the cells and subjected to reverse transcription reaction with M-MLV reverse transcriptase (Promega). Quantification of the cDNA was performed with SYBR master mixture on the Biorad machine. The qPCR primer sequence was as followed: ANP32E forward, 5’-TGCCTGTGTGTCAATGGGG-3’, and reverse, 5’-GCAGAGCTTCTACTGTACTGAGA-3’; and GAPDH forward, 5’-TGACTTCAACAGCGACACCCA-3’, and reverse, 5’-CACCCTGTTGCTGTAGCCAAA-3. The expression ANP32E was normalized to GAPDH.
Total protein was extracted from PANC1 and MIA cells with RIPA lysis buffer (Beyotime). BCA experiment (Beyotime) was conducted to measure the protein concentration. Equal amount of the protein was separated on 10-12% SDS-PAGE gels. Subsequently, the proteins were transferred onto PVDF membranes. 5% skim milk was used to block the membranes for 1 hour at room temperature. The membranes were incubated with indicated primary antibodies at 4℃ overnight. Antibody against ANP32E was from Abcam. β-actin primary antibody and the secondary antibodies were obtained from SantaCruz.
Cell proliferation was determined by CCK8 assay. Briefly, ANP32E silenced or over-expressed and its control cells were seeded into 96-well plates at the density of 2000 cells per well. The cells were cultured for 4 days. 1, 2, 3 and 4 days later, 10% the CCK8 regent (YEASEN) was added into each well and the plates were maintained at 37oC. 3 hours later, the OD value at 450 nm was measured on the micro-plate machine. The cell viability was normalized to the OD value of the first day.
Colony formation assay
A total of 500 shCtrl, shANP32E#1 and shANP32E#2, OE-Ctrl and OE-ANP32E PANC1 and MIA cells were seeded into the 6-well plates. 10 days later, colonies were formed and the cell culture was removed. After washed by PBS for three times, the colonies were fixed by methanol and stained by crystal violet. Subsequently, the plates were washed by clean water and dried at room temperature. Then the colonies were photographed by the camera.
Cell cycle analysis
PI staining was used to analyze the cell cycle in PANC1 and MIA cells. Indicated cells were seeded in triplicate in 6-well plates. 2 days later, the cells were re-suspended in FBS free culture medium and fixed by 70% ethanol. After stained by PI regent, flow cytometer was used to analyze the cell cycle distribution in PANC1 and MIA cells.
A total of 3×104 PANC1 or MIA cells in 200ul FBS free culture medium were seeded onto the upper surface of 8.0-μm filter migration chambers. The lower compartment contained 500ul DMEM medium with 10% FBS. 24 hours later, the cells attached on the upper surface were removed using cotton tips. The cells on the lower surface were fixed by methanol and stained by crystal violet. The images were photographed under a microscope.
Graphpad prism software was used to analyze the data as shown in the Figure. Students’t tests were used to determine the difference between two groups. One-way ANOVA was used to determine the difference when more than two groups. Statistical difference was considered significant when p<0.05.