Patients and clinical samples
Tumor and paired adjacent normal tissues were obtained from PC patients treated at Ruijin Hospital of Shanghai Jiaotong University of China. For quantitative polymerase chain reaction (qPCR) analysis, specimens were minced and stored in RNAlater (ThermoFisher Scientific, Waltham, MA, USA) for the isolation of total RNA. For western blot and immunohistochemical analyses, protein was isolated from specimens by freezing them in liquid nitrogen or fixing them in 4% paraformaldehyde, respectively. Institutional Review Committee of the Ruijin Hospital of Shanghai Jiaotong University of China approved current investigation in accordance with the guidelines of Helsinki Declaration.
Survival Analysis With Kaplan – Meier Plotter Web Tool
The web-based Kaplan-Meier Plotter (http://kmplot.com/analysis/index.php?p=service) was employed to determine PC patient five-year survival rate. Kaplan-Meier Plotter performed survival analyses based on gene expression levels.
Cell Lines And Cell Culture
The human pancreatic ductal epithelial cell line (HPDEC1) and PC cell lines (HPDEC1, CFPAC1, HPAC, PanC-1 and BxPC-3) were gained from ATCC. HPAC and PanC-1 cell lines were cultured in DMEM (Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum (HyClone, Logan, UT). CFPAC-1 cells were grown in Iscove’s modified Dulbecco’s medium (Invitrogen). BxPC-3 cells were cultured in RPMI-1640 Medium (Invitrogen). HPDECs were cultured in keratinocyte serum-free medium (Gibco, Grand Island, NY, USA) containing EGF (1 ng/ml) and BPE (50 mg/ml). The cell lines were maintained at 37°C in humid incubator with 5% CO2.
Vectors, Retroviral Infection, And Transfection
siRNA sequences were designed and chemically synthesized by QIAGEN. The following RRP9 siRNA sequences were used: siRNA#1: sense: 5’- AAUAAGGAGGAUAAGAGUGUC-3’, antisense: 5’- CACUCUUAUCCUCCUUAUUUA-3’; and siRNA#2: sense: 5’- UAAAUAAGGAGGAUAAGAGUG-3’, antisense: 5’- CUCUUAUCCUCCUUAUUUAAG-3’. The following IGF2BP1 siRNA: sense: 5’- AUGUAAAGCUUGUUCAUGGUG-3’, antisense:
5’-CCAUGAACAAGCUUUACAUCG-3’. Control siRNA: sense: 5’
-UUCUCCGAUCGUGUGACGU-3’, antisense: 5’-ACGUCACACGAUCGGAGAA-3’. Cells were transfected with siRNAs (50 nM) using Lipofectamine 3000 reagent (Invitrogen).
For RRP9 overexpression, cDNA encoding homo sapiens RRP9 (GenBank accession no. NM_004704.5) was prepared by PCR, sequenced, and separately cloned into pcDNA3.1 lentiviral expression vector (ThermoFisher Scientific). For infection, cells were grown to 70–80% confluence in 12-well plates, which were infected with lentiviral particles and polybrene. GFP-lentiviral particles acted as controls. We collected cells 48 h post-infection and processed for other assays.
Rna Extraction, Reverse Transcription (Rt), And Real-time Pcr
Total RNA was extracted from PC tissues and cell lines employing TRIzol (Life Technologies, Waltham, MA, USA). We reversed transcribed total mRNA through PrimeScript RT Reagent kit (TaKaRa, Kyoto, Japan). We carried out cDNA amplification and quantification using Bio-Rad CFX qRT-PCR detection system (Applied Biosystems Inc., Foster City, CA, USA) with SYBR Green Master (ROX; Roche, Toronto, ON, Canada). GAPDH was utilized as housekeeping gene, and 2−ΔΔCt method was applied to calculate relative gene expression values.
Western Blotting Analysis
We extracted protein from cell and tissue samples by RIPA buffer. Protein quantification was carried out applying BCA Protein Assay kit (Beyotime Institute of Biotechnology, Shanghai, China). We resolved equal quantities of protein through SDS-PAGE, which was transferred to PVDF membranes. After blocking with 5% BSA for two hours, membranes were incubated over the night at 4°C with following primary antibodies: anti-RRP9 (1: 500, Eterlife), anti-cleaved caspase-3 (1: 500, Abcam), anti-cleaved poly(ADP-ribose) polymerase (PARP) (1: 1000, Abcam), anti-p-AKT (Ser473) (1:2000, Cell Signaling Technology), anti-AKT1 (1:1000, Cell Signaling Technology), anti-p-BAD (Ser136) (1:500, Cell Signaling Technology), anti-BAD (1:1000, Cell Signaling Technology), anti-p-caspase-9 (Ser 196) (1:500, Abcam), anti-caspase-9 (1:1000, Abcam), anti-γ-H2AX (1:5000, Abcam) and anti-IGF2BP1 (1:100, Santa Cruz). Next, we incubated membranes for 1 h at room temperature with horseradish peroxidase-conjugated secondary antibodies (goat anti-rabbit/mouse, PIERCE, Waltham, MA, USA). After stripping, the membrane was re-probed with the loading control antibody, anti-GAPDH (1:1000; Santa Cruz). We visualized protein bands via chemiluminescence that enhanced.
Mtt Cell Viability Assay
PC cell sensitivities to gemcitabine exposure was assessed through MTT assay. Briefly, cells (2×103) plated onto 96-well plates were cultured overnight at 37°C, which were treated by varying gemcitabine concentrations for 1 d. Next, we incubated cells with MTT (0.5 mg/ml, Sigma) for 4 h at 37°C. We removed culture medium and added DMSO (150 µl) to each well (Sigma). Falcon microplate reader (BD-Labware) was utilized to measure the absorbance at 540 nm.
Colony Formation Assay
Briefly, we exposed cells to gemcitabine for 72 h, which were seeded into 24-well plates (8 ×102 cells per plate). Cell cultures were incubated for 10 days at 37°C in humid incubator with 5% CO2. After fixing, we stained colonies employing 0.2% crystal violet, and counted the colony number.
Apoptosis was examined applying an annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) kit (Abcam). Cells (1×106) were plated in 10-cm plates and treated with gemcitabine for 24 h. We harvested cells, washed them with PBS and re-suspended them in binding buffer (100 µL). Our team incubated samples with annexin V-FITC and PI for 15 min in dark, which were analyzed via FACS (Beckman Coulter, Pasadena, CA, USA).
The co-localization of IGF2BP1 and RRP9 was examined by immunofluorescence staining of PanC-1 or BxPC-3 cells that had been transfected with either the control vector or RRP9-OE plasmid. DNA damage was detected by immunofluorescence staining of γ-H2AX. Briefly, we cultured cells (2×105) on glass coverslips that placed in 24-well plates, which were exposed to gemcitabine for 24 h. After fixing with 4% formaldehyde for 15 min, we permeabilized samples with 1% Triton X-100 for 20 min, which were washed with PBS, blocked in 5% BSA for 30 min, then incubated with the following primary antibodies: anti-RRP9 (1: 100, Eterlife), anti-IGF2BP1 (1:50, Santa Cruz) or anti-γ-H2AX (1:250, Abcam, Cambridge, MA, USA) at 4°C over the night. After washing with PBS twice, we incubated samples with Alexa Fluor either 488-labeled anti-rabbit IgG or 594-labeled anti-mouse secondary antibodies (Thermo, Waltham, MA) for 1 h. Our team stained nuclei with DAPI. Samples were visualized by laser scanning confocal microscopy.
Our group lysed cells with RIPA buffer including broad-spectrum protease inhibitors. We incubated protein (1 mg) with 3 µg anti-RRP9 IgG and anti-IGF2BP1 IgG antibodies overnight at 4°C on a rotator. We added protein A agarose beads (Santa Cruz Biotechnology), and incubated samples for a further 2 h at 4°C. We washed agarose beads and extracted proteins that immunoprecipitated, which were subjected to western blot analysis. RRP9 and IGF2BP1 combination was predicted through Starbase (http://starbase.sysu.edu.cn/index.php).
Our group cultured cells (1×104) in 48-well plates for 1 d. Transfection of control or AKT-luciferase (AKT-luc) reporter plasmids (100 ng) and pRL-TK renilla plasmid (1 ng) was carried out applying Lipofectamine 3000 (Invitrogen). Dual Luciferase Reporter Assay Kit (Promega) was utilized to detect luciferase signals.
Immunohistochemical Staining (Ihc)
Human PC tissues, paired adjacent normal tissues and xenografts from nude mice were formalin-fixed and paraffin-embedded. We prepared tissue sections, which we stained using primary antibody against RRP9 (1: 400, Eterlife). The staining intensity was scored as follows: 0 (no staining); 1 (light yellow), 2 (yellow brown), and 3 (brown). Then, a value for the staining index (SI) was obtained by multiplying the positively-stained tumor cell percentages by staining intensity.
Subcutaneous Xenograft Tumor Model
A subcutaneous tumor model was established by randomly dividing BALB/c nude mice into 4 groups (n = 6/group). We subcutaneously injected mice in the left dorsal flank with PanC-1 cells transfected with either i) PanC-1/Vector, ii) PanC-1/RRP9-OE, iii) PanC-1/siRNA-Vector or iv) PanC-1/RRP9-siRNA#1 (2×106 cells/mouse). When the diameter of the xenografts reached 0.5 cm, mice were administered vehicle (Control) or gemcitabine (100 mg/kg) intraperitoneally twice a week for 41 d. The PanC-1/RRP9-OE group was treated with gemcitabine plus control or gemcitabine plus the AKT inhibitor MK-2206 (120 mg/kg body weight, 3 times/week) for 41 days. The tumor length and width were detected to evaluate tumor growth. Tumor volume was calculated by (L × W2)/2. An IVIS imaging system was used to monitor the tumors. At the end of experiments, we euthanized animals, removed and weighed their tumors. Formalin-fixed paraffin-embedded samples were prepared. The level of apoptosis in the paraffin-embedded tissue sections was determined by TUNEL assay kit (Promega). Institutional Animal Care and Use Committee of Ruijin Hospital affiliated to Shanghai Jiaotong University approved experimental procedures.
We performed statistical analysis using SPSS 11.0 statistical package with following tests: Fisher’s exact test, Chi-square test, log-rank test and Student’s 2-tailed t test. Multivariate statistical analysis was carried out with Cox regression model. Data are denoted by mean ± standard deviation (SD). P < 0.05 was considered as statistical significance.