BioProject

We performed a transcriptome analysis to determine what genes were altered in expression when U0126, MAP2K inhibitor, was administered to cells of human pancreatic cancer cell lines. More...

We performed a transcriptome analysis to determine what genes were altered in expression when U0126, MAP2K inhibitor, was administered to cells of human pancreatic cancer cell lines. Five genes including ETV5, ETV4, COL13A1, HIST1H4L/H4C13, and WDR62 were found to be particularly downregulated by the inhibition of MAPK pathway, meaning that these genes were preferentially upregulated by the active MAPK. The expression of ETV5 was most modulated, suggesting that ETV5 is strongly associated with the MAPK activity and may play a role in human pancreatic cancer. Overall design: Human pancreatic cancer cell lines AsPC-1, MIA PaCa-2, and PCI-35 were used in this study. AsPC-1 and MIA PaCa-2 were obtained from the American Type Culture Collec-tion (Manassas, VA, USA), and PCI-35 was provided by Dr. Hiroshi Ishikura [Int J Cancer 1993;54:972-977]. Unless otherwise stated, AsPC-1 and PCI-35 were cultured with RPMI-1640 with 10% fetal bovine serum, and MIAPaCa-2 was cultured with Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum. Cells of the human pancreatic cancer cell lines were seeded at 5 × 105 cells per dish in 10-cm culture dishes and cultured under 37°C in 5% CO2. After 24 hours, the medium was replaced with medium containing U0126 (Sigma-Aldrich, St. Louis, MO, USA), a MAP2K inhibitor, dissolved in dimethyl sulfoxide (DMSO) to a final concentration of 10 μmol/L, or with medium containing only the same amount of DMSO without U0126 as a control. After incubation for 24 hours, cells were dissociated for RNA isolation. Total RNA was isolated using the RNeasy Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instruction. The isolated total RNA was used for construction of a fragment library using TruSeq Stranded Total RNA LT Sample Prep Kit Gold (Illumina, San Diego, CA, USA). Constructed libraries were subjected to total transcriptome enrichment using a NovaSeq 6000 S4 Reagent Kit. The prepared transcriptome libraries were sequenced on an Illumina NovaSeq 6000 platform using the paired-end sequencing method. All procedures were performed according to the manufacturer’s instructions and done by Macrogen Japan Corp. (Tokyo, Japan). Reads were aligned to GRCh38 using HISAT2. Around one hundred million reads were mapped per sample. Known genes and transcripts were assembled with StringTie based on the reference genome model. The FPKM value of known genes obtained through StringTie analysis was used to detect the differentially expressed genes. During data preprocessing, low-quality transcripts were filtered out. Afterward, log2 transformation of FPKM+1 and quantile normalization were performed. Genes showing two-fold or more changes in expression by MAPK attenuation were interpreted as significant. Less...