Bioinformatics analysis
The RNA-sep data and a and corresponding clinicopathologic profiles of gastric cancer tissues samples and normal controls samples were obtained from The Cancer Genome Atlas (TCGA) database (http://gepia.cancer-pku.cn/index.html), and applied for analyzing the expression of RNPC1 in 408 gastric cancer tissues and 211 normal tissues.
Cell culture and transfection
The human gastric normal epithelial mucosa cell line (GES-1) and gastric cancer cell lines (MGC-823, MGC-803, SGC-7901) was provided by Prof. Daming Gao from the Shanghai Institute of Biochemistry and Cell biology, the Chinese Academy of Sciences. The MGC-823, MGC-803, SGC-7901were cultured in RPMI 1640 medium (Gibco, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, USA), 1% penicillin–streptomycin (Gibco, USA) solution in an incubator with humidified 5% CO2 at 37°C; while the GES-1 was cultured in DMEM medium (Gibco, USA); .
The gastric cancer cell lines were transfected with RNPC1 knockdown lentivirus (termed as shRNPC1-1,shRNPC1-2),a scrambled control (termed as SCR), RNPC1 overexpress lentivirus termed as RNPC1) or a negative control (termed as NC); which performed as described previously[43]. The cells were seeded into 6-wells plates and infected with the lentivirus, when the cells were at 30% confluence, 5 μg/ml polybrene was added into the 6-wells plates to enhance the infection efficiency. Then puromycin (3 μg/ml) was used to select the stable transfected gastric cancer cells. Western blotanalysis and quantitative real-time polymerase chain reaction (qRT-PCR) were applied to determine the efficiency of transfection.
The AURKB was knockdown with small interfering RNAs (siRNA) (genpharma, China) in gastric cancer cell lines.The siRNA sequences was shown in Supplementary Table 2. The cells were transfected with about 10 nm siRNA or controls with lipofectamine 2000 transfection reagent (Invitrogen, USA) for 24h according to the manufacturer's instruction.
Western blot
Western blot analysis was performed as described previously[43].The primary antibodies used were RNPC1 (Santa Cruz, USA, sc365898), aurora b(abcam, USA, ab45145), cyclin A2(abcam, USA, ab181591), cyclin D1(abcam, USA, ab205718) , cyclin E1(abcam, USA, ab71535). the secondary antibodies were purchased from cell signaling technology. The intensity of the bands was determined using densitometric analysis. β-actin antibody (Abcam,USA) was used to as control for normalization.
RNA extraction, reverse transcription and qRT-PCR
Total RNA was extracted from cells using Trizol reagent (Invitrogen, USA) and cDNA was synthesized using Primescript RT Reagent (TaKaRa, China) following manufacturer's instructions. The qRT-PCR was performed on StepOne Plus Real-Time PCR system (Applied Biosystems, USA). β-actin was amplified as an internal standard. Each sample was replicated three times and the data analyzed using the 2-ΔΔCT method.The detail PCR primers used were shown in Supplementary table 2.
CCK-8 assay
The 5000 gastric cancer transfected cells were seeded into 96-well plates. Cell proliferation was documented at 0, 24, 48, 72 and 96 h after seeding. When measured the cell proliferation, the medium in each well was replaced with 100 μl fresh medium containing 10% CCK-8, and the cells were incubated for 1 h. The absorbance was measured by microplate reader (Biotek-Eon, Biotech, USA) at 450 nm.
Colony formation assay
The transfected gastric cancer cells (1000 cells/well) were seeded into six-well plates and incubated in 1640 medium with 10% FBS. After about 2 weeks, colonies were fixed with methanol for 10 min, and then stained with 0.1% crystal violet (Beyotime, China). The colonies were imaged and counted.
EdU assays
The proliferation of RNPC1 overexpressed and knockdown gartric cancer cell lines were evaluated with the BeyoClick™ EdU cell proliferation kit with Alexa Fluor 555 (#C0075S, Beyotime, Shanghai, China) according to the manufacturer’s protocol. The cells were seeded into six-well plates and incubated overnight with 5% CO2 at 37℃.Then 1ml EdU (20 μM) was added to the culture medium then incubated for 2 hours.The cells were fixed with 4% paraformaldehyde for 15 minutes, the washed with PBS containing 3% BSA for 3 times.The PBS with 0.3% Triton X-100 PBS used
to permeabilize the nuclear membrane for 15 minutes at room temperature. Finally, cells were stained using a BeyoClick™ EdU cell proliferation kit with Alexa Fluor 488(#C0071S, Beyotime, Shanghai, China) according to the manufacturer’s protocol.
Wound healing assay
The transfected gastric cancer cells were cultured in 6-well plates until 100% confluence. The cell layer was scratched using a 200-μl pipette tip and the loose cells were washed away with phosphate-buffered saline (PBS, pH 7.4), and the old media was replaced with the fresh media. Images of wound healing were taken after 24 h. The distance of the wound was measured in 6 random selected microscopic fields (100×) for each condition and time point (0, 24 h).
Transwell assays
The transwell assays were performed with transwell chamber (Millipore, NY, USA). For cell invasion assays, BD MatrigelTM basement membrane matrix was added to the upper chamber for one night at 37°C. In transwell assays with or without Matrigel, 5×104 cells cells were re-suspended in serum-free medium and added into top compartment, and medium containing 10% FBS was added to the bottom compartment. After 24 h, the methanol and crystal violet were used for fixation and stained of cells. The number of migrating and invading cells was counted under 200× magnification(Olympus,Japan).
Cell cycle analysis
Cells were collected, and fixed in ethanol at -20°C for overnight. Then cells stained with propidium iodide (PI) by the cycletest plus DNA reagent kit (BD Biosciences, USA) at room temperature for 30 min and then measured by flow cytometry (Guava, BD Biosciences, USA).The ratios of cells in the G1, S, G2 phases were analyzed.
Tumorigenesis in nude mice
Female BALB/C nude mice (4-6 weeks old, 18-22g) were obtained from Model Animal Research Center of Nanjing University (Nanjing, China). All mice were feeding in the animal facility. The animal studies were performed in accordance with the institutional ethics guidelines for animal experiments, which was approved by the animal management committee of Nanjing Medical University.The mice were randomly allocated into two groups and injected with SGC-7901-shRNPC1 cells and SGC-7901-SCR cells (1 × 107 cells), respectively. The tumor volume (V) was monitored by measuring the width (W) and length (L) with a caliper every 2 days and the tumor volume was calculated using the formula V=(W2×L)/2. The mice were sacrificed after 3 weeks and checked for final tumor weight.
Actinomycin D assay
The stably transfected gastric cancer cell lines were treated with 5 µg/ml actinomyclin D (Act D) for 0, 1, 2, 4, 6, 8 h. Total RNAs were harvested, and then subjected to qRT-PCR analysis. The relative quantification of AURKB transcript was calculated by the 2−ΔΔCt method and normalized based on β-actin. Then the relative half-life of AURKB was calculated.
RNA immunoprecipitation (RIP)
The transfected gastric cancer cell lines were lysed with RNA immunoprecipitation lysis buffer (Millipore, USA) and then incubated with 5 µg of rabbit polyclonal anti-RNPC1 (Santa Cruz Biotechnology, USA) or nonimmunized rabbit IgG at 4°C overnight. The RNA-protein immunocomplexes were brought down by protein A/G magnetic beads, followed by RNA purification. After that, the purified RNA was subjected to RT-PCR and qRT-PCR. The primers to detect human AURKB, IgG and human antigen R (HuR) mRNA expressions were the same as those described previously[43].
RNA electrophoretic mobility shift assay (REMSA)
The recombinant RNPC1 proteins were expressed and purified as previously describe41,42. To generate REMSA probes, regions in AURKB mRNA 3′-UTR was amplified by PCR with primers including T7 (5′-TAATACGACTCACTATAGGG-3′). The primers for AURKB probes were showed in Supplementary table 2. Biolabelled RNA probes were made from in vitro transcription with a MEGA shortscript Kit (Ambion, Waltham, MA, USA) in the presence of biotin-16-UTP (Roche) according to the manufacturer′s instruction.The REMSA was performed as previously with some modification(27,40) with the LightShift Chemiluminescent RNA EMSA Kit (Thermo, Waltham, MA, USA).
Dual-Luciferase reporter assay
The RNPC1 overexpress (RNPC1) and the control (NC) gastric cancer cells were cultured in the 24 well plates. Then cells were cotransfected with 5 ng plasmids that containing AURKB 3′-UTR, AURKB-mutant 3′-UTR or empty vector with Lipofectamine3000 (Invitrogen, USA). We mutated all U residues to G in the AURKB-mutant 3′-UTR . Twenty-four hours after transfection, luciferase activity was measured with the dual luciferase kit according to manufacturer’s procedure (Promega, USA). The fold change in relative luciferase activity is a ratio of the luciferase activity induced by RNPC1 divided by that induced by NC. The sequence of the AURKB 3′-UTR was same as the REMSA.
IC50 determination
The cells were seeded in 96-well platewith5000 cells/well, and incubated for 24h. Then the cells were treated with increasing concertrations of AURKB inhibitors AZD1152-HQPA or hesperadin(MedChem Express,shanghai, china ) for 48h.The cell viability was determined with CCK-8 assay and the concentration that induced 50% growth inhibition (IC50) were calculated.
IHC staining
Eighty-five gastric cancer tissues and 26 tumor-adjacent normal tissues were obtained from gastric cancer patients who underwent resection surgery at the Cancer Hospital of Shantou University Medical College, Shantou, Guangdong,China. This study was approved by the Ethics Committee Cancer Hospital of the Cancer Hospital of Shantou University Medical College. The specimens were assessed by immunohistochemistry (IHC) and the diagnosis was verified by histopathological examination. IHC staining was performed according to the procedure described previously[43].The same tissue samples were stained with the monoclonal antibodies against RNPC1 ( Santa Cruz) and aurora b(abcam, USA). The images were observed under 100 × magnifications under a microscope(Olympus, japan).
Immunofluorescence
The immunofluorescence were performed with immunol fluorence staining kit (Beyotime, China) according to the manufacturer's protocol. The cells were plated in the laser copolymerized petri dish, and fixed with immunol staining fix solution. After being permeabilized with 0.1 % triton X-100 and blocked with immunol staining blocking buffer, the cells were incubated with primary antibody (anti-α-tubulin antibodies 1:1000, Sigma-Aldrich, USA; anti-γ-tubulin 1:50, abcam, USA) at 4°C overnight. Following incubated with rabbit goat anti-rabbit IgG-FITC or mouse goat anti-mouse IgG-FITC (Cell Signaling Technology, 1:600) in the dark for 1 h and counterstained with 46-diamidino-2-phenylindole (DAPI) for 10 min. Fluorescent images were captured by a confocal laser microscope (Olympus, Japan).
Statistical analysis
Statistical analysis was performed with SPSS 20 software and GraphPad Prism 6. The differences between two independent groups were analyzed using Student’s t test. A P-value< 0.05 was considered to represent a statistically significant difference.