Long noncoding RNA signatures induced by TLR7 and type I IFN signaling in activated human plasmacytoid dendritic cells

Using genome-wide sequencing approaches we viewed the contributions of Toll-like receptor 7 (TLR7) and type I interferon (IFN-I) in the regulation of coding and noncoding RNA expression in CAL-1 pDC treated with R848 or IFNβ. Functional enrichment analysis revealed both the unique and synergistic roles of TLR7 and IFN-I signaling in the orchestration of pDC function. Total RNA-seq profiling was performed on CAL-1 cells (1 million cells/mL) were cultured in 0.1% FBS CAL-1 media in triplicate in a 12- well tissue culture plate for 6 hours prior to stimulation with 1,000 U/mL recombinant human IFN-β (PBL Interferon Source) or 1ug/mL R848 (Invitrogen) or 1ug/mL R848 (Invitrogen) + 1ug/mL recombinant vaccinia virus B18R protein (Thermo Fisher Scientific) or mock stimulation. Total RNA was harvested 12 hours post stimulation

本研究采用全基因组测序策略，探究了Toll样受体7（Toll-like receptor 7, TLR7）与I型干扰素（type I interferon, IFN-I）在经R848或IFNβ处理的CAL-1浆细胞样树突状细胞（plasmacytoid dendritic cell, pDC）中对编码RNA与非编码RNA表达的调控作用。功能富集分析结果显示，TLR7与IFN-I信号通路在调控pDC功能的过程中兼具独特作用与协同效应。本研究对CAL-1细胞开展总RNA测序（RNA-seq）分析：将CAL-1细胞以1×10^6个/mL的密度接种于含0.1%胎牛血清（fetal bovine serum, FBS）的CAL-1培养基中，于12孔组织培养板中设置3次生物学重复，培养6小时后分别采用以下试剂进行刺激：1000 U/mL重组人IFN-β（PBL Interferon Source）、1 μg/mL R848（Invitrogen）、1 μg/mL R848（Invitrogen）联合1 μg/mL重组痘病毒B18R蛋白（Thermo Fisher Scientific），以及空白刺激。最终于刺激后12小时收集总RNA。