Materials
Dulbecco’s modified Eagle’s medium (DMEM) was obtained from Hyclone (Catalog Number: SH30243.01B+, Logan, Utah, USA). Fetal bovine serum (FBS) was obtained from Biowest (Catalog Number: S1810, Loire Valley, France). The lentiviral expression vector (pFH-L) and packaging vectors (pVSVG-I and pCMVΔR8.92) were purchased from Hollybio (Shanghai, China). Lipofectamine 2000 and TRIzol® Reagent was purchased from Invitrogen (Carlsbad, CA, USA). M-MLV Reverse transcriptase was purchased from Promega (Madison, WI, USA). All other chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA).
Analysis of oncomine data
To determine the expression pattern of ZFR in pancreatic cancer, two datasets, including Badea Pancreas [15] and Segara Pancreas [16] in Oncomine database (www.oncomine.org) were used. The gene expression of ZFR was compared between pancreatic cancer tissues with normal pancreatic tissues according to the standard procedures as previously described [17].
Cell culture
Human pancreatic cancer cell line, PANC-1 and human embryonic kidney cell line 293T were obtained from the Cell Bank of Chinese Academy of Science (Shanghai, China). Both cell lines were cultured in DMEM supplemented with 10 % FBS at 37 °C with 5 % CO2.
Construction of recombinant lentiviral vector
The cDNA sequence of ZFR was obtained from Gen Bank (NM_016107). The shRNA target sequence for ZFR was 5′-GCCAAGGTGCAACTCAGTATACTCGAGTATACTGAGTTGCACCTTGGCTTTTT-3′, which was subjected to BLAST analysis against the human genome database to eliminate cross-silence phenomena with nontarget genes. A scrambled fragment (5′-GCGGAGGGTTTGAAAGAATATCTCGAGATATTCTTTCAAACCCTCCGCTTTTTT-3′) that has no significant homology with human gene sequences was used as a negative control. DNA oligonucleotides to produce plasmid-based shRNA were cloned into the pFH-L vector by use of NheI/PacI restriction sites. The lentiviral expression vector (pFH-L) and packaging vectors (pVSVG-I and pCMVΔR8.92) were cotransfected into 293T cells with Lipofectamine 2000 according to the manufacturer’s instructions. The supernatant was collected 48 h later, centrifuged (4000g, 4 °C, 10 min) to remove cell debris, filtered through 0.45-μm cellulose acetate filters, and then concentrated again (4000g, 4 °C, 15 min). The lentiviral vectors expressed green fluorescence protein (GFP), which allowed for titering and measurement of their infection efficiency in transduced cells. PANC-1 cells were dispensed into 6-well plates at a density of 50,000 cells per well and transduced with shRNA-expressing lentivirus at a multiplicity of infection (MOI) of 75. GFP expression was observed by fluorescent microscopy 4 days post-transduction.
Quantitative real-time polymerase chain reaction (qRT-PCR)
Cells transduced with shRNA-expressing lentivirus were divided into two groups (shCon, shZFR). PANC-1 cells were harvested after lentivirus transduction for 6 days. Total cellular RNA was extracted using Trizol reagent and reversely transcripted to cDNA by M-MLV reverse transcriptase according to the manufacturer’s instructions. qRT-PCR products were detected with SYBR Green on BioRad Connect Real-Time PCR platform following the procedure: denaturation at 95 °C for 1 min, 40 cycles of denaturation at 95 °C for 5 s and extension at 60 °C for 20 s. Specific cDNAs were then amplified by qRT-PCR using the following primers: ZFR, 5′-TCCCAATGCTAAGGAGATGC-3′ (forward) and 5′-TTCTTCTCGTCTTCGCCAGT-3′ (reverse); CDK2, 5′-TCCAGGATGTGACCAAGCC-3′ (forward) and 5′-CTGAGTCCAAATAGCCCAAGG-3′ (reverse); CyclinA, 5′-GTTCCTCCTTGGAAAGCAAAC-3′ (forward), 5′-GGGCATCTTCACGCTCTATTT-3′ (reverse); CyclinD1, 5′-GCCCTCGGTGTCCTACTTC-3′ (forward), 5′-CCTCCTCGCACTTCTGTTCC-3′ (reverse); p27, 5′-GCTACCCTTGACAAGAAAAGAC-3′ (forward), 5′-AAAGTATGCTTACTAAAGGTCCTG-3′ (reverse) and β-actin, 5′-GTGGACATCCGCAAAGAC-3′ (forward) and 5′-AAAGGGTGTAACGCAACTA-3′ (reverse). The β-actin was used as internal control. Relative quantitation was analyzed by taking the difference ΔC(T) between the C(T) of β-actin and C(T) of ZFR and computing 2-ΔΔC(T) as described previously [18].
Western blot analysis
Cells transduced with shRNA-expressing lentivirus were divided into two groups (shCon, shZFR). PANC-1 cells were harvested after lentivirus transduction for 6 days. Total protein was extracted with 2 × SDS Sample Buffer [100 mM Tris–HCl (pH 6.8), 10 mM EDTA, 4 % SDS, 10 % Glycine]. Total protein concentration was determined by BCA assay. Protein extracts were separated by 10 % SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5 % nonfat dry milk in Tris-buffered saline with Tween 20 (TBST) for 1 h at 37 °C, and then incubated overnight at 4 °C in TBST with primary antibody, including rabbit anti-ZFR (1:1000, SAB2104153, sigma), mouse anti-CDK4 (1:500, #2906, cell signaling), rabbit anti-CDK2 (1:1000, #2546, cell signaling) and rabbit anti-GAPDH (1:100000, 10494-1-AP, Santa cruz). Following incubation with horseradish peroxidase-conjugated goat anti-rabbit/mouse secondary antibody for 1 h, the membranes were detected using enhanced chemiluminescence (ECL) kit (Amersham) and visualized by exposure to X-ray film. GAPDH was used as a control to verify equal protein loading.
MTT assay
To detect cell viability, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT) colorimetric assay was performed 4 days after lentivirus transduction. Briefly, PANC-1 cells were dispensed into 96-well plates at a density of 2000 per well. The plates were incubated for one to 5 days at 37 °C. On each day, 100 ml of MTT (5 mg/ml) was added and incubated for 4 h. Afterwards, the entire supernatant was discarded and acidic isopropanol (10 % SDS, 5 % isopropanol and 0.01 mol/L HCl) was added at a volume of 100 μl per well and incubated at 37 °C for 10 min. The absorbance at 595 nm of each well was determined using an ELISA reader.
Colony formation assay
To detect cell proliferation, colony formation assay was performed 4 days after lentivirus transduction. Briefly, PANC-1 cells were dispensed into 6-well plates at a density of 500 per well. The culture medium was changed at three-day intervals. PANC-1 cells were cultured for 10 days until the most single colony contains more than 50 cells. The colonies were stained with crystal violet for 15 min, and then washed with water and air-dried. Cell colonies were captured and counted under a microscope.
Fluorescence-activated cell sorting analysis (FACS)
To detect cell cycle progression, flow cytometry assay was performed 7 days after lentivirus transduction as described previously [19]. Briefly, PANC-1 cells were dispensed into 6-cm dishes at a density of 200,000 per dish. After culture at 37 °C for 40 h, cells were harvested, fixed in 70 % ethanol, and stored overnight at 4 °C. The cells were then treated with NaCl/Pi staining solution (50 µg/mL PI and 100 µg/mL RNase A). Following incubation for 1 h in the dark at room temperature, cells were analyzed by flow cytometry (FACSCalibur; Becton–Dickinson, San Jose, CA, USA). The fractions of the cells in G0/G1, S and G2/M phases were analyzed with dedicated software (Becton–Dickinson).
Cell migration and invasion assays
The migration and invasion ability of PANC-1 following ZFR knockdown were evaluated using Transwell (8 µm pore size, Millipore). Briefly, cells were suspended in serum-free medium and seeded into Transwell inserts either uncoated (for migration assay) or coated (for invasion assay) with growth factor-reduced Matrigel (BD Biosciences, Bedford, MA). Botton wells were filled with complete medium. Then the invaded cells were fixed with methanol and stained with a crystal violet solution. Finally, the number of migratory and invasive cells were counted in five fandom fields under a microscope at 200 × magnification.
Statistical analysis
Statistical analysis was performed using SPSS 13.0 software package (SPSS Inc, Chicago, IL, USA). Data were expressed as mean ± standard deviation from three independent experiments. Statistical differences between the groups were compared using Student’s t test. A P value of less than 0.05 was considered statistically significant.
