Next Generation Sequencing Facilitates Quantitative Analysis of Macrophage (RAW264.7) with and without arctigenin treatment

Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to explore the mechanism and target of arctigenin affecting macrophages (RAW264.7) Methods: Total mRNA was extracted from cells using an RNA extraction kit (Qiagen K.K., Tokyo, Japan). RNA-Seq was performed using HiSeq X Ten for next-generation sequencing by OE Biotech. Co., Ltd. (Shanghai, China). Sequenced reads were mapped to the mm9 genome using hisat2.The RNA-Seq reads that were aligned against the exon regions of genes were quantified with HTSeq-Count in the RefSeq mm9 annotation. GSEA was performed on mRNA expression datasets using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Gene signatures were considered enriched if the false discovery rate (FDR) q-values and the family-wise error rate (FWER) p-values show significant difference. Result: The GSEA from the KEGG database indicated that AG-induced biological processes involved several inflammatory process. GSEA methods on immunologic gene sets of Molecular Signatures Database show that the gene set of NFAT5 knockout passed the filtering criteria (p<0.01) Conclusion: NFAT5 was the target of arctigenin Overall design: Macrophage (RAW264.7) with vector, with 0.5 mg/mL LPS alone or combined 1 µM arctigenin treatment.