Human cell lines and tissues
Five pancreatic cancer cell lines (AsPC-1, BxPC-3, CFPAC-1, Panc-1, and Pau8988) and human pancreatic ductal epithelial (HPNE) cells were purchased from the Cell Repository, Chinese Academy of Sciences (Shanghai, China). In addition, 293T cells were obtained from Shanghai Institute of Hematology. Panc-1, Patu8988, HPNE, and 293T cells were cultured in DMEM. AsPC-1 and BxPC-3 cells were grown in RPMI-1640. CFPAC-1 cells were grown in Iscove's modified Dulbecco's medium. All media contained 10% inactivated FBS (Gibco, Carlsbad, CA, USA), 1 × 105 U/L penicillin, and 100 mg/L streptomycin (Gibco). Cells were cultured in a humidified atmosphere containing 5% CO2 at 37 °C. Seventy-two pairs of pancreatic cancer tissues and adjacent normal pancreatic tissues were collected at Ruijin Hospital affiliated with the Shanghai Jiaotong University School of Medicine (Shanghai, China). All 72 cancer samples were histologically identified as adenocarcinoma. All tissue samples were snap-frozen in liquid nitrogen and stored at -80 °C until use. All enrolled patients met the following criteria: (1) pathological diagnosis of pancreatic cancer, (2) complete clinicopathological and follow-up data, and (3) no preoperative chemotherapy. Written informed consent was obtained from all patients, and the study protocol was approved by the ethics committee of Ruijin Hospital.
RNA extraction and quantitative real-time PCR (qRT-PCR)
TRIzol reagent (Invitrogen, Carlsbad, CA) was used to extract total RNA, according to the manufacturer’s instructions. Cytoplasmic and nuclear RNA was extracted using the PARIS kit (Invitrogen). RNA samples were analyzed using NanoPhotomentN120(IMPLEN, Germany) to detect the concentrations and values of A260/A280 and A260/A230. CDNA was synthesized from 1 µg RNA using the Evo M-MLV RT Kit with the gDNA Clean for qPCR II kit (Accurate Biology, Hunan, China), according to the manufacturer’s instructions. QRT-PCR assays of mRNA expression levels were performed using SYBR® Green Premix Pro Taq HS qPCR Kit II (Accurate Biology, Hunan, China) on qTOWER3 84G (Analytik Jena AG, Jena, Germany) according to the manufacturer’s instructions. The housekeeping genes U6 and glycerinaldehyde-3-phosphat-dehydrogenase (GAPDH) were used as reference genes. Primer sequences are listed in Table 1.
Western blot analysis
Cells were lysed in RIPA buffer (Beyotime, Shanghai, China) supplemented with a protease inhibitor cocktail (MCN Biotech, Suzhou, China), loaded, and separated on a 10% SDS-PAGE gel (EpiZyme, Shanghai, China). The samples were transferred onto polyvinylidene fluoride membranes and incubated with the following primary antibodies: anti-KLF12, anti-Cyclin D3, anti-CDK4, anti-CD44, anti-Sox2, anti-NANOG, anti-ALD1H1, anti-Oct4, anti-p-pb, anti-p21, anti-p-AKT, anti-AKT, anti-GAPDH (Table 2). GAPDH was used as the controls.
Subcutaneous tumor tissues from nude mice were fixed in 4% paraformaldehyde, dehydrated, and embedded in paraffin. The antigens were retrieved, and nonspecific binding was blocked using 4% normal goat serum (Gibco). Subsequently, cell coverslips were incubated with anti-PCNA, anti-CD44, and anti-CD133 primary antibodies (Table 3), followed by incubation with HRP-conjugated goat anti-rabbit IgG antibodies (1:1000, Cell Signaling, MA, USA). Then, 3,3-diaminobenzidine chromogen substrate solution was used to visualize the results.
For in vitro experiments, miR-7155-5p mimics, negative control (NC) mimics, miR-7155-5p inhibitor, and NC inhibitor were synthesized by Tsingke (Beijing, China). Two shRNA-GFP vectors with two shRNA sequences targeting the 3’-UTR of LINC01137 and a NC shRNA-GFP vector were synthesized by Bioegene (Shanghai, China). Full-length LINC01137 and KLF12 cDNA was synthesized and inserted into a lentiviral vector or plasmid (Bioegene, Shanghai, China). BxPC-3, CFPAC-1, and PANC-1 cells transduced with the lentivirus were treated with 2 μg/mL puromycin for 48 h to establish stable cell lines. All transfections were performed using Lipofectamine 3000 (Invitrogen). The cells were collected 48 h post-transfection. The miRNA mimics and inhibitor, shRNA, and NC sequences are listed in Table 4.
Sphere formation assay
Pancreatic CSC (PCSC) spheres were generated by culturing primary pancreatic cancer cells (2000-4,000 cells/ml) in ultra-low attachment plates (Corning) in FBS-free DMEM/F12 (Invitrogen) supplemented with B27 1:50 (Invitrogen), 20 ng/mL bFGF (PAN-Biotech), and 50 U/mL penicillin/streptomycin (Thermo Fisher Scientific). Seven days later, the spheres were harvested, trypsinized into single cells, and re-cultured for subsequent assays. Seven days later, the spheres were measured, counted, and photographed under a Zeiss Axio Vert A1 microscope (Zeiss, Oberkochen, Germany).
Cell proliferation assay
A CCK-8 kit was purchased from Meilune Bio (Dalian, China). The cells were plated in 96-well plates and treated for 24 h. Then, the CCK-8 reagent was added to the cells for another 4 h. Absorbance was measured using a microplate reader at 450 nm. A BeyoClick™ EdU Cell Proliferation Kit with Alexa Fluor 555 was purchased from Beyotime (Shanghai, China). The cells were plated in 12-well plates, cultured for 24 h, and then fixed using 4% paraformaldehyde and stained with DAPI after incubation with 50 mM EdU solution for 2 h. EdU-labeled cells were photographed under a Zeiss Axio Vert A1 microscope (Zeiss, Oberkochen, Germany).
Colony formation assay
The cells (1000-2000 cells/well) were seeded into 6-well plates and incubated with complete medium at 37 °C for 2–3 weeks. Then, the cells were fixed with 4% paraformaldehyde and stained with 2% crystal violet. Images were obtained, and the number of colonies was counted.
Flow cytometry (FCM)
Apoptosis rates were determined using FCM after staining with Annexin-V-7AAD and APC. The Annexin-V-7AAD and APC Apoptosis Detection Kit was purchased from BioLegend (San Diego, CA). The cells were plated in 6-well plates and treated as described in the Results section. Then, the cells were harvested, washed twice, resuspended in binding buffer, and stained with Annexin-V-7AAD and APC solution for 15 min at room temperature. Finally, the samples were subjected to FCM. After seeding in 6-well plates for 48 h, the cells were washed three times with cold PBS. Then, the supernatant was discarded via centrifugation, and the cells were resuspended at 1 × 106 cells/ml in 75% ethanol. After fixation at 4 ℃ for 8 h, cold ethanol was discarded via centrifugation, and the cells were washed with PBS twice. After removal of the supernatant, the cells were stained with 300 μl propidium iodide (Sigma, St. Louis, MO) for 30 min at 37 ℃ in a dark environment. CytoFLEX 5 (Beckman Coulter, Fullerton, CA) was used to record red fluorescence at an excitation wavelength of 488 nm to analyze the cell cycle. CD44 and CD133 antibodies were purchased from BioLegend (San Diego, CA) and used according to the manufacturer’s protocol. CytoFLEX 5 (Beckman Coulter, Fullerton, CA) was applied to record APC-Cy7 and PE panel signals to analyze CD44 and CD133 status.
RNA fluorescence in situ hybridization (FISH)
Cy3-labeled LINC01137 probes were purchased from RiboBio (Guangzhou, China). BxPC-3, CFPAC-1, and Panc-1 cells were fixed with 4% formaldehyde and permeabilized with 0.5% TritonX-100. The cells were then hybridized with Cy3-labeled probes. The nuclei were stained with DAPI. Images were acquired using a Zeiss LSM900 (Zeiss, Oberkochen, Germany).
Luciferase reporter assays
Wild-type (WT) and mutant (MUT) 3-UTRs of LINC01137 and KLF12 reporter plasmids were constructed through BioGene (Shanghai, China). Then, 293T cells were co-transfected with luciferase constructs and miR-7155-5p mimics, according to the manufacturer’s protocol. After transfection for 48 h, luciferase activity was measured using a dual-luciferase reporter assay kit (Vazyme). Each experiment was conducted in triplicates.
In vivo tumorigenicity model
Animal experiments were conducted with the approval of the Animal Ethics Committee of Ruijin Hospital, affiliated with the Shanghai Jiaotong University School of Medicine (Shanghai, China). The pancreatic cancer cells were digested and suspended in cold PBS at a density of 108 cells/ml. Approximately 100 μl of the cell suspension containing 107 cells was injected subcutaneously into 4–5-week-old male BALB/c nude mice on the right and left sides of the armpit. After almost a month, the tumors were harvested and analyzed.
In vivo limiting dilution assays
Pancreatic cancer cells were digested and suspended in cold PBS at a density of 107–104 cells/ml at four concentrations. Approximately 100 μl of cell suspension, containing 106–103 cells, was injected subcutaneously into 4–5-week-old male BALB/c nude mice on the right and left sides of the armpit. After almost a month, the tumors were harvested and analyzed. CSC frequency assay was followed by previous research(Hu & Smyth,2009).
The gene expression profiles and related clinical data of patients were retrieved and downloaded from The Cancer Genome Atlas (TCGA) database. Dataset GSE51971 was retrieved and downloaded from Gene Expression Omnibus (GEO) database. Dataset MTAB6690 was downloaded from ArrayExpress database. The Box plot and prognosis analysis about data from TCGA were plotted by GEPIA (http://gepia.cancer-pku.cn/index.html). The heatmap was plotted by http://www.bioinformatics.com.cn, a free online platform for data analysis and visualization. The potential subcellular localization of LINC01137 was predicted on lncLocator(Lin et al.,2021b). The microRNAs that are interacting with LINC01137 were predicted using DIANA(Paraskevopoulou et al.,2016), miRDB(Chen & Wang,2020), and LNCSNP2 (http://bioinfo.life.hust.edu.cn/lncRNASNP#!/) datasets. The mRNAs that bind to miR-7155-5p were predicted through miRDB(Chen & Wang,2020), miRWalk(Sticht et al.,2018), miRPathDB(Kehl et al.,2020), and TargetScan (http://www.targetscan.org/vert_71/) datasets.
Statistical analyses were performed using SPSS 20.0 and GraphPad Prism 8.0. Experiments were repeated independently at least three times, and the results are presented as the means ± standard deviation (SD). One-way analysis of variance, Student’s t-test, and chi-squared test were used to analyze the differences between different groups. Survival curves were analyzed using the Kaplan–Meier method, and log-rank tests were used to evaluate differences between the groups. Statistical significance was set at p < 0.05.