The Ethics Committee of Shanghai Tenth People’s Hospital, Tongji University School of Medicine ratified our study. Written informed consents were acquired from patients before their participation in this study. All experimental methods abided by the Declaration of Helsinki. All animal studies were undertaken in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals issued by US National Institutes of Health.
Clinical sample collection
Cancer and adjacent normal tissues (more than 2 cm away from tumor margins) were surgically acquired from 28 patients (18 males, 10 females; at the age of 39–72 years with a mean age of 54 years) who were pathologically confirmed PC at Shanghai Tenth People's Hospital, Tongji University School of Medicine from May 2016 to December 2017. The patients enrolled had not received either local or systemic treatment prior to the operation. There were nine cases at the stage I, five cases at the stage II and 14 cases at the stage III. The specimens were assessed histopathologically by the hospital pathology department with detailed clinical data were recorded. All specimens were frozen in liquid nitrogen in a quick manner and stored at controlled temperature of − 80 °C for later analysis.
One normal human pancreatic ductal epithelial cell line HPDE6-C7 (HZ-H296; Shanghai Huzhen Biotechnology Co., Ltd., Shanghai, China) and three PC cell lines BxPC-3, MIAPaCa-2, and PANC-1 (ATCC, Manassas, VA, USA; www.atcc.org) were used. These cells were subjected to culture with the RPMI 1640 medium (consisted of 10% FBS, 100 U/mL streptomycin and 100 U/mL penicillin) at controlled temperature of 37 °C under 5% CO2, with the medium being renewed every 2 days. Upon growing to 80–90% confluence, cells were passaged, and exponentially growing cells were used for subsequent experiments. The expression of NORAD in cell lines was tested by RT-qPCR; the PANC-1 cells had the highest NORAD expression and were consequently selected for further study.
Sequences for NORAD, miR-202-5p and ANP32E were obtained from the NCBI, and Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. (Shanghai, China) was entrusted with the construction of plasmids including miR-202-5p mimic, small interfering RNA (si)-ANP32E, ANP32E, si-NORAD, NORAD, and corresponding negative controls (NCs) by using pCMV-Flag-N/C plasmid vector.
The third generation of cells were trypsinized and seeded in plates with 24 wells to form monolayer cells. The cells were divided into two parts and subjected to transfection using Lipofectamine 2000 reagent. One portion of the cells was transduced using miR-202-5p mimic, si-ANP32E and ANP32E, alone or in combination. The other portion of cells was treated with si-NORAD, NORAD, miR-202-5p mimic, either alone or in combination. Finally, after 48-h transfection, to screen stably-transfected cells, cells were maintained for 4 weeks under standard condition in G418 (1000–2000 μg/mL) medium, which was renewed every 3–5 days.
Bioinformatics prediction and dual-luciferase reporter assay
WT luciferase reporter plasmid ANP32E (ANP32E-WT-Luc) containing WT ANP32E sequence and the MUT luciferase reporter plasmid ANP32E (ANP32E-Mut-Luc) containing MUT ANP32E sequence were all from Shanghai Genechem Co., Ltd. (Shanghai, China). Thereafter, 293T cells underwent cotransfection with miR-202-5p mimic or miR-202-5p mimic-NC and reporter vectors with the use of Lipofectamine™ 2000. After incubation for 24 h, luciferase activity was assessed at 560 nm by Dual-Luciferase Reporter Assay Kit and a microplate reader (Thermo MK3).
WT and MUT primers of NORAD were designed and synthesized by Sangon. Total RNA content of PANC-1 cells was extracted and amplified by means of PCR with WT and MUT primers. Hind III and Bgl II enzyme endonuclease sites were added at both ends of the amplified products. pGL3-Basic luciferase reporter vector (Promega) was then digested by restriction endonuclease Hind III/Bgl II, and the large fragments were recovered by electrophoresis. Ligase 4 was linked the amplified target gene and vector to obtain NORAD-WT-Luc and NORAD-MUT-Luc plasmids, which were subsequently transformed into the E. coli competence sequence. After colony identification by PCR, the plasmids were extracted from the colony shaker kit containing the target fragment and sequenced. Other procedures were the same as described above.
RNA-pull down assay
Cells were subjected to transfection with the use of 50 nM biotin-labeled Bio-miR-202-5p-WT and Bio-miR-202-5p-MUT. The cell lysates were incubated with RNase-free BSA and yeast tRNA precoated Dynabeads Streptavidin Magnetic Beads. The enrichment of NORAD was measured by RT-qPCR.
PANC-1 cells were initially lysed in RIP lysis solution and centrifuged at 14,000 rpm at 4 °C for 10 min. The supernatant was harvested, a portion of which was removed as input while the other was probed with antibodies against rabbit anti-human Ago2 (ab186733, 1: 50, Abcam) and rabbit anti-human IgG (ab109489, 1: 100, Abcam, taken as NC), with SNRNP70 (Millipore) used as a positive control, for co-precipitation. At last, the immunoprecipitated RNA was isolated and analyzed by means of RT-qPCR.
Fluorescence in situ hybridization (FISH)
Cells post 24-h transfection in each group were detached with trypsin by shaking for 5 min, and then centrifuged for 2–3 min in 1.5 mL Eppendorf (EP) tubes. The cells were fully mixed with the pre-cooled CER I and lysed on ice for 10 min. Additional spinning was conducted with precooled CER II, followed by incubation and centrifugation. Next, the supernatant containing cytoplasmic components was transferred into a new EP tube and then stored at − 80 °C for later analysis. Afterwards, the supernatant was spun with pre-cooled nuclear extraction reagents and incubated. Then, it was remixed for 15 s at intervals of 10 min and finally centrifuged for 10 min at 4 °C. Finally, the supernatant containing the nuclear fraction was put into fresh EP tubes, and stored at − 80 °C.
The coverslips were dried and fixed, after which cells were subsequently treated with protease K, DEPC-4% paraformaldehyde, then acetic acid, and incubated with 200 μL pre-hybridization solution for 1 h at room temperature. After that treatment, 250 μL hybridization solution containing 0.1–0.2 ng/μL probe was added for a further incubation at 65 °C for 14 h. The cells were washed, sealed and finally incubated with anti-DIG-AP Fab antibody (diluted 1:5000 din Buffer B2) overnight at 4 °C. Thereafter, the coverslips were developed with freshly prepared BCIP/NBT solution for 3–24 h in the dark.
Total RNA content of PC cells was extracted by TRIzol reagent, and the purity and concentration of the extracted RNA were determined by NanoDrop ND-1000. Thereafter, cDNA was synthesized with a PrimeScript RT reagent Kit, while RNA was converted to cDNA by a One Step PrimeScript MicroRNA Gene Synthesis Kit. RT-qPCR of the product was implemented using a Quanti-Tect SYBR Green PCR kit on the ABI7500 quantitative PCR system. With U6 and GAPDH serving as the internal reference, relative expression pattern of each target gene was measured by means of 2−ΔΔCt method. PCR primer sequences are listed in Additional file 1: Table S1.
Western blot analysis
After 72 h of transfection, total protein was extracted and the concentration was assessed by a bicinchoninic acid kit. All the protein lysates were separated using 10% SDS-PAGE, transferred onto a polyvinylidene fluoride membrane, and sealed by 5% skimmed milk powder. After that, the membrane underwent overnight probing at 4 °C with primary antibodies, namely rabbit anti-human antibodies to cleaved-caspase 3 (1:1000, #9665, Cell Signaling Technology, Beverly, MA, USA), cleaved-caspase 9 (1:1000, #9508, Cell Signaling Technology), PARP1 (1:1000, ab32064, Abcam), Oct4 (1:1000, ab181557, Abcam), Nanog (1:200, ab21624, Abcam), and Sox2 (1:1000, #14962, Cell Signaling Technology). Thereafter, the membrane underwent re-probing with HRP-conjugated secondary goat anti-rabbit for 1 h at 37 °C. Finally, the membrane was visualized with enhanced chemiluminescence reagent (Pierce). Ratio of the gray value of target bands to that of the internal reference GAPDH (1:2500, ab9485, Abcam) band represents the relative protein expression.
Cells post 24-h transfection were resuspended in Aldefluor buffer to adjust the density to 1 × 106 cells/mL. The activity of aldehyde dehydrogenase (ALDH), a stem cell marker, was detected by an Aldefluor kit according to the instructions. The cells were subjected to incubation at 37 °C for 25 min with 15 μM ALDH specific inhibitor exogenous 4-(diethylamino)benzaldehyde (DEAB) and 0.15 μM ALDH substrate. Then the activity of ALDH was measured by a flow cytometer.
Exponentially growing cells were cultured with 20 μL of MTT (5 mg/mL) at 0, 12, 24, 48 and 72 h after transfection for 4 h in the dark. Cells of each well were supplemented with 150 μL dimethylsulfoxide and placed on a shaking table for 10 min, and the OD value was then measured by means of a microplate reader (DG5031, Shanghai Kehuai Instruments Co., Ltd., Shanghai, China) at 490 nm.
After 24 h of transfection, cells were detached with trypsin without EDTA and centrifuged. After that, collected cells were fixed by addition of 3 mL pre-cooled 70% ethanol, centrifuged, and stained with 0.5 mg/mL propidium iodide (PI) staining solution, followed by detection by a flow cytometer at more than 575 nm. Apoptosis rate of PC cells was assessed by an Annexin V-FITC/PI double staining kit (556547, Shanghai Solja Technology Co., Ltd., China). After centrifugation, cells were resuspended in pre-cooled 1× phosphate buffer saline, centrifuged at 200 rpm for 5–10 min and resuspended in 300 µL 1× binding buffer. Next, the cells were incubated with 5 µL of Annexin V-FITC and stained with 5 µL PI, followed by analysis with a flow cytometer (Cube6, Sysmex Partec, Am Flugplatz, Görlitz, Germany). FITC was detected at 480 and 530 nm, while PI at a wavelength greater than 575 nm. The proportion of stem cell markers CD24+ and CD44+ cells was then calculated. Cells were incubated with FITC-conjugated CD44 (mouse anti-human, BD Biosciences, 555478), and phycoerythrin (PE)-conjugated CD24 (mouse anti-human, BD Biosciences, 555428), along with their corresponding isotype controls (BD Biosciences, 555742 and 55554) for cell surface staining, washed twice with the use of PBS, and resuspended in PBS for analysis/sorting.
Colony formation assay
PC cells post 24-h transfection were detached with 0.25% trypsin and triturated into single cell suspension. This single-cell suspension was plated in plates with 6 wells (1 × 104 cells/mL) and grown for 2 weeks under standard condition. When cell colonies were observed by naked eye, the culture was halted and the cells underwent 3.7% methanol fixation for 10 min and 0.1% crystal violet staining for 10–30 min. After staining and washing, the cells were photographed and the number of clones (> 50 cells) per well was counted using the Image J software for statistical analysis .
Sphere formation assay
Cells post 24-h transfection were plated in ultralow attachment plates with 24 wells at a density of 1000 cells/well in serum-free DMEM/F-12 medium containing B27 (1:50), 20 ng/mL basic fibroblast growth factor, and 20 ng/mL epidermal growth factor. The number of microspheres formed within 7 days was counted, and the colony formation ratio was calculated based on a factor of 1000.
Xenograft tumors in nude mice
BALB/c mice aged 5 weeks (equal numbers of male and female) were randomly grouped into 13 groups (12 for each group). The mice were housed under room temperature conditions at a stable humidity of 50–60% under a 12-h light/dark cycle with free access to drinking water. For tumor propagation analysis, 1.5 × 106 cells resuspended in 0.1 mL serum-free DMEM was mixed with 0.1 mL Matrigel and injected subcutaneously into the back of nude mice. After 3 days, a second cell suspension of the same volume was injected at the same site. Tumor formation and volume were observed every 2 days after injection. Four weeks later, mice were euthanized, thus at 5 weeks after tumor innoculation. The weight and volume of the tumors were measured. The volume was then calculated as the calculation (length × width2)/2.
The measurement data described as mean ± standard deviation and SPSS 21.0 software was used to analyze the data. The statistical significance was measured using paired t-test, unpaired t-test, one-way ANOVA with Tukey’s multiple comparisons test and two-way ANOVA or repeated measures ANOVA, followed by Bonferroni post hoc test for multiple comparisons. A value of p < 0.05 denotes statistical significance.