Sun Yulong, Kuang Yuanyuan, Zuo Zhuo
School of Life Sciences, Northwestern Polytechnical University, Xi'an 710072, China.
Key Laboratory for Space Biosciences & Biotechnology, Institute of Special Environmental Biophysics, School of Life Sciences, Northwestern Polytechnical University, Xi'an 710072, China.
Genes (Basel). 2021 May 9;12(5):705. doi: 10.3390/genes12050705.
Neuropeptide FF (NPFF) is a neuropeptide that regulates various biological activities. Currently, the regulation of NPFF on the immune system is an emerging field. However, the influence of NPFF on the transcriptome of primary macrophages has not been fully elucidated. In this study, the effect of NPFF on the transcriptome of mouse bone marrow-derived macrophages (BMDMs) was explored by RNA sequencing, bioinformatics, and molecular simulation. BMDMs were treated with 1 nM NPFF for 18 h, followed by RNA sequencing. Differentially expressed genes (DEGs) were obtained, followed by GO, KEGG, and PPI analysis. A total of eight qPCR-validated DEGs were selected as hub genes. Subsequently, the three-dimensional (3-D) structures of the eight hub proteins were constructed by Modeller and Rosetta. Next, the molecular dynamics (MD)-optimized 3-D structure of hub protein was acquired with Gromacs. Finally, the binding modes between NPFF and hub proteins were studied by Rosetta. A total of 2655 DEGs were obtained (up-regulated 1442 vs. down-regulated 1213), and enrichment analysis showed that NPFF extensively regulates multiple functional pathways mediated by BMDMs. Moreover, the 3-D structure of the hub protein was obtained after MD-optimization. Finally, the docking modes of NPFF-hub proteins were predicted. Besides, NPFFR2 was expressed on the cell membrane of BMDMs, and NPFF 1 nM significantly activated NPFFR2 protein expression. In summary, instead of significantly inhibiting the expression of the immune-related gene transcriptome of RAW 264.7 cells, NPFF simultaneously up-regulated and down-regulated the gene expression profile of a large number of BMDMs, hinting that NPFF may profoundly affect a variety of cellular processes dominated by BMDMs. Our work provides transcriptomics clues for exploring the influence of NPFF on the physiological functions of BMDMs.
神经肽FF（NPFF）是一种调节多种生物活性的神经肽。目前，NPFF对免疫系统的调节是一个新兴领域。然而，NPFF对原代巨噬细胞转录组的影响尚未完全阐明。在本研究中，通过RNA测序、生物信息学和分子模拟探索了NPFF对小鼠骨髓来源巨噬细胞（BMDM）转录组的影响。用1 nM NPFF处理BMDM 18小时，然后进行RNA测序。获得差异表达基因（DEG），随后进行GO、KEGG和PPI分析。总共选择了8个经qPCR验证的DEG作为枢纽基因。随后，通过Modeller和Rosetta构建了8个枢纽蛋白的三维（3-D）结构。接下来，用Gromacs获得枢纽蛋白经分子动力学（MD）优化的3-D结构。最后，通过Rosetta研究了NPFF与枢纽蛋白之间的结合模式。共获得2655个DEG（上调1442个，下调1213个），富集分析表明NPFF广泛调节由BMDM介导的多种功能途径。此外，经MD优化后获得了枢纽蛋白的3-D结构。最后，预测了NPFF-枢纽蛋白的对接模式。此外，NPFFR2在BMDM的细胞膜上表达，1 nM NPFF显著激活NPFFR2蛋白表达。总之，NPFF并没有显著抑制RAW 264.7细胞免疫相关基因转录组的表达，而是同时上调和下调了大量BMDM的基因表达谱，这表明NPFF可能会深刻影响由BMDM主导的各种细胞过程。我们的工作为探索NPFF对BMDM生理功能的影响提供了转录组学线索。
