Ethics statement and subjects
In total, 80 patients diagnosed with PC who were operated in the First Affiliated Hospital of Fujian Medical University between 2009 and 2020 were included in this study. The clinicopathological characteristics including age, sex, alcohol history, smoking history, CEA, CA199, tumor size, tumor stage, lymph node invasion, and metastasis were recorded. The current study was approved by the Ethics Committee of the First Affiliated Hospital of Fujian Medical University (Fujian, China).
Cell lines and cell culture conditions
All of the cell lines used in this study were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA), including the cancerous AsPC-1, CFPAC-1, Capan-1, SW1990, PANC-1, SW1990, BxPC-3, and MIA-PaCa-2 cell lines and the non-cancerous HPDE6C7 cell line. All of the above-mentioned cells were cultured in 5% CO2 at 37°C in 100% humidity in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% antibiotic/antifungal solution (Biowest, Nuaillé, France).
Immunohistochemistry
All of the tissues were fixed with 10% formalin for at least 48 h and paraffin-embedded for immunohistochemistry (IHC) assays, as described previously [10]. We incubated tissue sections with primary antibodies (Table 1) at 4°C overnight and treated them with an UltraVision Quanto Detection System Horseradish Peroxidase (HRP) 3,3′-Diaminobenzidine (DAB) Kit (Thermo Scientific [Thermo Fisher Scientific, Waltham, MA, USA]) as per the manufacturer’s instructions. Expression changes were identified as follows: 10 highly magnified fields were randomly selected for counting under an Olympus CX23 microscope (Olympus Corp., Tokyo, Japan) at 200× resolution, and cancer cells were counted under the microscope as previously described [10].
Western blot
Total protein was extracted from homogenized tissues or cells, and the lysate was mixed with hydrolysis buffer. We determined the protein concentration using the bicinchoninic acid (BCA) method. Bromophenol blue and β-mercaptoethanol were added into the sample buffer before electrophoresis. After separation by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), the proteins were transferred to a polyvinylidene fluoride membrane (Bio-Rad Laboratories, Hercules, CA, USA). The membrane was incubated with primary antibody (Table S1) at 4°C overnight. After incubation for 2 h in secondary antibody, the protein bands were visualized using enhanced chemiluminescence (ECL) substrate and photographed. ImageJ software version 1.8.0 (National Institutes of Health, Bethesda, MD, USA) was used to examine relative expression of proteins. Each experiment was repeated in triplicate.
Quantitative reverse-transcriptase PCR
Total RNA was extracted from selected cells or tissues using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) as per the manufacturer’s instructions. We examined the RNA integrity and purity using agarose gel electrophoresis. We polyadenylated the total RNA using poly(A) polymerase (Ambion, Austin, TX, USA), as described previously [11]. Total poly(A)-tailed RNA, reverse-transcription primers (Table S2), and ImPro-II reverse transcriptase (Promega, Fitchburg, WI, USA) were used for reverse transcription per the manufacturer’s instructions. For quantitative reverse-transcriptase PCR (qRT-PCR), we used Fast Start Universal SYBR Green Master Mix (Roche Diagnostics GmbH, Mannheim, Germany).
Colony formation and holoclone sphere formation assays
For the colony formation assay, first, the cells were cultured for 14 days in 6-well plates at 5 × 102 cells per well. Then, the cells were fixed by methanol/acetic acid (3:1, v/v) and stained with 0.5% crystal violet (Sigma-Aldrich, St. Louis, MO, USA). The resulting cells were counted using a microscope (Olympus IX81).
For the holoclone assay, 100 cells were cultured for several days in a 6-well dish, and then the number of holoclones was counted. Cloning efficiency was defined as the percentage of cells that established a holoclone.
Flow cytometry
The cells were suspended in 1 × 106 cells/ml, and 5 μL Annexin V and propidium iodide (PI) staining solution were added to 300 μL of the cell suspension. Stained cells were assayed by a FACSort Flow Cytometer (BD, San Jose, CA, USA) after incubation at room temperature for 10–15 min.
RNA immunoprecipitation assay
A Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore, USA) was used for the RNA immunoprecipitation (RIP) assay according to the manufacturer’s instructions. Briefly, cell extracts were immunoprecipitated with Sepharose bead-conjugated antibodies against CELF2 or IgG at 4°C for 6 h. To remove proteins from the complex, 0.1% SDS/Proteinase K (0.5 mg/mL, 30 min at 55°C) was used. Immunoprecipitated proteins and RNAs were detected using western blot and qRT-PCR, respectively.
Transcriptome sequencing
Total RNA was isolated and purified using TRIzol (Life, cat. 265709, CA, USA) following the manufacturer’s procedure. After inspecting the quality of the total RNA using an Agilent 2100 Bioanalyzer (Agilent, cat. G2939AA, CA, USA) and a NanoPhotometer® (Implen, cat. N60, Munich, Germany), mRNA with poly(A) was purified from 1 μg total RNA using VAHTS® mRNA Capture Beads with Oligo(dT) (Vazyme, cat. N401-01, Nanjing, China) through two rounds of purification. Subsequently, mRNA fragments were interrupted using the VAHTS® Universal V6 RNA-seq Library Prep Kit (Vazyme, cat. NR604, Nanjing, China) at 94°C for 8 min and reversed transcribed into cDNA, which was used to synthesize U-labeled double-stranded DNA. An A-base was added to the blunt ends of each strand to ligate the indexed adapters which contain a T-base at the tail end. After UDG enzyme treatment of the U-labeled double-stranded DNA, size selection was performed with VAHTS® DNA Clean Beads (Vazyme, cat. N411, Nanjing, China). Then the ligated products were amplified with PCR under the following conditions: initial denaturation at 98°C for 5 min; 12–17 cycles of denaturation at 98°C for 10 sec, annealing at 60°C for 30 sec, and extension at 72°C for 30 sec; final extension at 72°C for 5 min. The average insert size of the cDNA library was 280±80 bp. After purification by VAHTS® DNA Clean Beads (Vazyme, cat. N411-02, Nanjing, China), quality control of concentration and fragment size was performed using an Agilent 2100 Bioanalyzer (Agilent, cat. G2939AA, CA, USA) and Qubit assay tubes (Life, cat. 1604220, CA, USA). At last, we performed the 2 × 150-bp paired-end sequencing (PE150) on an Illumina NovaSeq™ 6000 system (Illumina Corporation, San Diego, USA) following the vendor’s recommended protocol by Guangzhou Huayin Health Medical Group CO., Ltd. (Guangzhou, China). The mRNA expression levels were calculated using RNA-Seq by Expectation Maximization (RSEM) (v1.3.1) and normalized to fragments per kilobase per million reads (FPKM) values. The differentially expressed mRNAs were screened using the edgeR package (https://bioconductor.org/packages/release/bioc/html/edgeR.html) in R. Genes with |log2(fold change)| ≥ 1, and P < 0.05 was considered to be significantly differentially expressed. Subsequently, GO and KEGG pathway enrichment analyses were conducted using the clusterprofiler package in R. GSEA_Linux_4.0.3 (https://www.gsea-msigdb.org/gsea/index.jsp) was used to complete the enrichment analysis of the gene set with default parameters using the expression file obtained by RSEM as input.
Overexpression plasmid and siRNA transfection
We purchased the overexpression plasmidand negative controls (NCs) of CELF2 from GenePharma (Shanghai, China). Cells were seeded into 6-well plates at a concentration of 5 × 105 cells/2 ml per well. After 48 h, cells reached 60% of confluence. We then transfected the cells with overexpression plasmidor NCs using Lipofectamine 3000 (Invitrogen) as per the manufacturer’s instructions at a final concentration of 50 nM. Small interfering RNAs (siRNAs) targeting CD44 were transfected into cells using the methods described above (Table S3). The final concentration of siRNA was 100 nM.
Cell migration and invasion assay
To detect cell migration and cell invasion, we used Transwell chambers (pore size, 8 μm; BD Biosciences, Franklin Lakes, NJ, USA). These chambers were coated with Matrigel (Corning Labware Products Inc., Corning, NY, USA) for cell invasion assays, and they were left uncoated for cell migration assays. For both types of assays, we seeded PC cells into the upper chamber of the Transwell and added medium containing serum to the bottom chamber. After 24 h, cells that had migrated into the Matrigel coating layer were fixed with paraformaldehyde and then stained with crystal violet to allow visualization. We observed, counted, and photographed the cells under an Olympus IX73 microscope (Olympus).
Lentivirus transduction
The lentivirus pEZX-MR04 plasmid expressing CELF2 and NCs were obtained from GeneCopoeia Inc. (Rockville, MD, USA). EndoFectin Lenti transfection reagent was used to co-transfect lentiviruses expressing CELF2 or the NC into HEK293T cells. After 48 h incubation, lentiviral particles were harvested by centrifugation (500 g for 10 min) and filtered. Cells were transduced using the lentivirus expressing CELF2 or NC. Stably transduced cells were selected for further study. Cells were cultured for 14 days in the presence of puromycin (2 μg/ml). The gene expression levels of CELF2 were measured using qRT-PCR.
In vivo assay to determine tumor growth
Male BALB/c nude mice (average age, 6 weeks) from the Shanghai Laboratory Animal Center were acquired for mouse experiments. All of the mouse protocols were reviewed and approved by the Fujian Medical University Ethics Review Committee. Cells stably transfected with the CELF2 overexpression lentivirus or empty vectors were subcutaneously injected into the hypochondriac regions of the selected mice. Tumor formation in these mice was observed for 5 weeks. Then the tumors were harvested for weight and volume measurements.
Statistical analysis
SPSS software version 21.0 (IBM Corp., Armonk, NY, USA) was used for statistical analysis. All of the data are reported as mean ± standard deviation (SD). Data were evaluated using Student’s t-test or one-way ANOVA. The χ2 test was used to evaluate correlations between expression levels and clinicopathological features. Pearson’s correlation coefficient was used for the correlation analysis. The Kaplan–Meier approach was used for survival analysis estimates. P < 0.05 was considered to be statistically significant.