Reagents and Chemicals
Fetal bovine serum (FBS) and Iscove’s modified Dulbecco’s medium (IMDM) were obtained from Gibco (USA). The CCK-8 assay kit, Annexin V-FITC assay kit, and Cell cycle assay kit were obtained from Thermo Fisher Scientific (USA). Primary antibodies against PDCD4 (1:500 dilution) and GAPDH (1:500 dilution) were purchased from Thermo Fisher Scientific (USA). HRP-conjugated goat anti-rabbit IgG (1:2000 dilution) and anti-mouse IgG (1:2000 dilution) were purchased from Invitrogen (USA). The EdU cell proliferation detection kit and antibiotics (100 μg/mL streptomycin and 100 U/mL penicillin) were purchased from Solarbio (Beijing, China). All other chemicals were of reagent grade.
Ethics Statement
The tissues of fifteen patients with PDAC (cases) and normal tissues (controls) were used. PDAC was diagnosed by histopathologic evaluation of biopsied pancreatic tissue or by endoscopy. The cancerous and para-cancerous tissues were collected before surgery or radiotherapy. The study was approved by the Biomedical Ethics Committee of the West China Hospital of Sichuan University and was conducted in accordance with the Declaration of Helsinki and its later amendments. All participants provided written informed consent prior to being enrolled in this study.
RNA Extraction and Library Construction
The PDAC tissue samples (15 tumor and 15 normal) were collected and extracted according to the QIAamp Viral RNA Mini Kit. Small RNA sequencing libraries were constructed using the Multiplex Small RNA Library Kit for Illumina® (NEB, USA). LncRNA sequencing libraries were constructed using Stranded RNA Library Kit for Illumina® (NEB, USA). The constructed libraries were sequenced by Illumina Novaseq 6000 (Whbioacme, Co., Ltd, Wuhan).
Data Analysis
Raw data in fastq format were processed with Perl scripts. Quality filtering of clean data through removing reads containing adapters, poly-N, too short reads and low-quality reads from raw data. At the same time, the number of clean reads, clean Q20, clean Q30, and GC contents of the clean data were calculated. All data analysis about miRNA, mRNA, lncRNA were based on clean data. Reference genome and gene annotation files were downloaded directly from the genome website (GRCh38.p13). The known miRNA sequences were obtained in miRBase. miRNA expression levels were obtained using miRDeep2. Differential expression analysis of cancer compared with normal tissues was performed using DESeq2. miRNA target prediction was performed by TargetScan, picTar, microT, miRmap, RNA22, PITA and miRanda. Differential expressed mRNA and lncRNA analysis of cancer compared with normal tissues was performed using DESeq2. The screening criteria for significantly different genes are corrected P-values of 0.05, and log2 (fold change) ≥1. The target mRNA and lncRNA prediction of different expression miRNA uses Starbase database (version 3.0; starbase.sysu.edu.cn/index.php), which provides the prediction results of seven miRNA databases (TargetScan, picTar, microT, miRmap, RNA22, PITA and miRanda). The target genes were compared with different expression genes, and selected the overlap genes. The candidate DEL (different expression lncRNA)-DEM (different expression miRNA)-DEG (different expression mRNA) ceRNA network using Cytoscape software (version 3.8.2).
KEGG/GO pathway enrichment analysis
The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis uses cluster-profiler of R package. According to the KEGG annotations of genes, selecting all genes of human as background, and using P<0.05 as the significance threshold to obtain statistically significant high-frequency annotations relative to the background. In addition, Gene Ontology (GO) can be divided into three parts: Molecular Function, Biological Process and Cellular Component. We selected the all known cancer-associated pathways according the previously study to construct the scatter diagram of KEGG and GO pathways.
Cell Culture
Human pancreatic cancer cells CFPAC-1 was purchased from the American Type Culture Collection (ATCC, USA). Cells were cultured in IMDM (Gibco, USA) supplemented with 10% FBS and antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin).
Inhibition and overexpression treatment
The miR-199a-5p sequence was retrieved from the miRBase database. The miR-199a-5p mimic, miR-199a-5p mimic NC, miR-199a-5p inhibitor, and miR-199a-5p inhibitor NC sequences were designed and synthesized by Ribobio (Guangzhou). To express PDCD4 and VASH1-AS1, a DNA fragment encoding the full-length PDCD4 and VASH1-AS1 were first amplified from the CFPAC-1 cell line (Table 1), and then cloned into pcDNA3.1 (Ribobio, Guangzhou) at the EcoR1 and Not1 sites, respectively.
Table 1 Mir-199a-5p mimics, inhibitor sequences, VASH1-AS1 and PDCD4 primer sequences
gene name
|
sequence
|
miR-199a-5p
|
5′-UAGCUUAUCAGACUGAUGUUGA-3′
|
miR-199a-5p reverse
|
5′-TCAACATCAGTCTGATAAGCTA-3′
|
miR-199a-5p mimic
|
5′-UAGCUUAUCAGACUGAUGUUGA-3′
|
3′-AUCGAAUAGUCUGACUACAACU-5′
|
miR-199a-5p mimic NC
|
5′-UUUGUACUACACAAAAGUACUG-3′
|
3′-AAACAUGAUGUGUUUUCAUGAC-5′
|
miR-199a-5p inhibitor
|
5′-UCAACAUCAGUCUGAUAAGCUA-3′
|
miR-199a-5p inhibitor NC
|
5′-CAGUACUUUUGUGUAGUACAAA-3′
|
PDCD4-Forward
|
5′-GAATTCTGGATGTAGAAAATGAGCAGA-3′
|
PDCD4-Reverse
|
5′-GCGGCCGCTCAGTAGCTCTCTGGTTTAAG-3′
|
VASH1-AS1-Forward
|
5′-GTCTCTTCCTTCCTAGGCC-3′
|
VASH1-AS1-Reverse
|
5′-TAGTGGTGCTTTTTAAATTTATTTTTAACTGTTT-3′
|
Vector constructions and Double Luciferase assay
The 3’-UTR of human PDCD4 and VASH1-AS1 containing the miR-199a-5p binding sites were cloned into pmiR-RB-REPORT vector (primers were listed in Table 2) (Ribobio, Guangzhou), which were named h-PDCD4-WT and h-VASH1-AS1-WT. Mutation in miR-199a-5p binding site was performed through QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA), with pmiR-RB-REPORT-WT as a template and named h-PDCD4-MUT and h-VASH1-AS1-MUT.
PDAC cells were seeded in a 96-well plate and incubated for 24 h before miRNA mimics or mimics NC were added. PDCD4 and VASH1-AS1 3’UTR Dual Luciferase Reporter vector or mutant vector were added to 15μL Opti-Mem medium and 25μL Lipofectamine 2000 (Invitrogen) for dilution. At 36 h after transfection, the fluorescence value was measured using the Dual Luciferase Reporter Assay System (Promega).
Table 2 PDCD4 and VASH1-AS1 3′-UTR amplification primers
primer name
|
sequence
|
PDCD4 3′-UTR-Forward
|
5′-TGTCTGACTGCCACTCCTTTC-3′
|
PDCD4 3′-UTR-Reverse
|
5′-AGCTGAGGTAATATGGGCTTG-3′
|
VASH1-AS1 3′-UTR-Forward
|
5′-TATGGATTCCTGCGGGTCAC-3′
|
VASH1-AS1 3′-UTR-Reverse
|
5′-CTGGCCTTGGGATGTGATGA-3′
|
Cell Transfection
CFPAC-1 cells (1×106 cells/mL) were seeded in a 6-well plate, and 2 mL of IMDM containing serum was added. The cells were placed in an incubator with a CO2 concentration of 5 % and cultured at 37 °C until the cell confluence reached 70-80%. Separately, miR-199a-5p mimics, mimics NC, miR-199a-5p inhibitors, inhibitor NC, PDCD4-sirna, PDCD4-overexpression vector, VASH1-AS1-sirna and VASH1-AS1-overexpression vector were added to 100 μL of Opti-MEM serum-free medium. After incubation for 48 h, the cells were collected for subsequent experiments.
Real-time PCR
Total RNA was reverse transcribed to cDNA using a Reverse Transcription Kit (Takara Co., Ltd., Dalian, China). The designed primers were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). cDNA was amplified using SYBR® Premix Ex Taq™ (TaKaRa, Dalian). Gene expression levels were calculated by the ΔΔCt method with GAPDH and U6 as internal controls for mRNA, lncRNA and miRNA respectively. The primer sequences used in this manuscript were listed in Table3.
Table3 QPCR primer sequences for miR-199a-5p, PDCD4, VASH1-AS1 and internal controls
gene name
|
sequence
|
miR-199a-5p
|
Forward 5′-TCCCCCAGTGTTCAGACTAC-3′
|
Reverse 5′-GCAGGGTCCGAGGTATTC-3′
|
U6
|
Forward5′-CGCTTCGGCAGCACATATAC-3′
|
Reverse5′-AAATATGGAACGCTTCACGA-3′
|
PDCD4
|
Forward5′-TTGGAGGGGAAGGCTAGTCA-3′
|
Reverse5′-AGCAATAAACTGGCCCACCA-3′
|
VASH1-AS1
|
Forward5′-TGGTCTAAGGAAGCTGGCAGGAG-3′
|
Reverse5′-ATGATGGTGTGAAGGGCAGGAAAC-3′
|
GAPDH
|
Forward5′-TCAAGAAGGTGGTGAAGCAGG-3′
|
Reverse5′-TCAAAGGTGGAGGAGTGGGT-3′
|
Western Blot Assay
Western blot assay in CFPAC-1 cells referred to the previous protocol [19], rabit-PDCD4 (1:500 dilution), rabbit GAPDH (1:200 dilution) and secondary antibody anti-rabbit IgG-HRP (1:1000 dilution) were used in our experiments. All results were expressed as the ratio of target protein to GAPDH.
CCK-8 Analysis
CCK-8 analysis in CFPAC-1 cells referred to the previous manuscript [20].
Flow Cytometry Analysis
Flow Cytometry Analysis in CFPAC-1 cells referred to the previous manuscript [19].
EdU Fluorescence Analysis
EdU Fluorescence Analysis in CFPAC-1 cells referred to the previous manuscript [21].
Statistical Analysis
SPSS software (version 22.0; SPSS, Chicago, USA) and GraphPad Prism 5.0 (GraphPad software, San Diego, California, USA) were used for data analysis. The data screening standard was three times the mean ± standard deviation. T-test was used to evaluate differences between groups, and a P value of <0.05 was regarded as to be statistically significant.