Gene expression profile of THP-1 monocytes following knockdown of DAP12, a causative gene for Nasu-Hakola disease

Nasu-Hakola disease (NHD), also designated polycystic lipomembranous osteodysplasia with sclerosing leukoencephalopathy (PLOSL), is a rare autosomal recessive disorder characterized by progressive presenile dementia and formation of multifocal bone cysts, caused by a loss-of-function mutation of DAP12 or TREM2. TREM2 and DAP12 constitute a receptor/adaptor complex expressed on osteoclasts, dendritic cells, macrophages, monocytes, and microglia. At present, the precise molecular mechanisms underlying development of leukoencephalopathy and bone cysts in NHD remain largely unknown. We established THP-1 human monocyte clones that stably express small interfering RNA (siRNA) targeting DAP12 for serving as a cellular model of NHD. Genome-wide transcriptome analysis identified a set of 22 genes consistently downregulated in DAP12 knockdown cells. They constituted the molecular network closely related to the network defined by cell-to-cell signaling and interaction, hematological system development and function, and inflammatory response, where NF-kappaB acts as a central regulator. These results suggest that a molecular defect of DAP12 in human monocytes deregulates the gene network pivotal for maintenance of myeloid cell function in NHD. The siRNA vector construct targeting the DAP12 sequence (SI) and the control vector construct targeting the scrambled sequence (SCR) were generated by using GeneClip U1 Hairpin cloning system (Promega). The vectors were transfected in THP-1 cells by using Lipofectamine LTX reagent. The stable cell lines were selected by incubating them for approximately two months in the feeding medium with inclusion of 200 microgram/ml Hygromycin B. Then, two SI clones named SI5 and SI17, in addition to two SCR clones named SCR1 and SCR4, were selected by limiting dilution of the cells in a manner of a single cell per well plated in a 96-well cell culture plate.

纳苏-哈科拉病（Nasu-Hakola disease, NHD）又称囊性脂膜性骨发育不良伴硬化性脑白质病（polycystic lipomembranous osteodysplasia with sclerosing leukoencephalopathy, PLOSL），是一种罕见的常染色体隐性遗传病，以进行性早老性痴呆及多灶性骨囊肿形成为特征，由DAP12或TREM2的功能丧失突变引发。TREM2与DAP12构成受体-接头蛋白复合物，在破骨细胞、树突状细胞、巨噬细胞、单核细胞及小胶质细胞中表达。目前，NHD患者脑白质病变与骨囊肿形成的确切分子机制仍未完全明确。我们构建了稳定表达靶向DAP12的小干扰RNA（small interfering RNA, siRNA）的THP-1人单核细胞克隆，以作为NHD的细胞模型。全基因组转录组分析鉴定出22个在DAP12敲低细胞中持续下调的基因。这些基因构成的分子网络与细胞间信号传导与相互作用、造血系统发育与功能、炎症反应相关的分子网络高度关联，其中核因子κB（NF-κB）作为核心调控因子。上述结果表明，人单核细胞中DAP12的分子缺陷会失调维持髓系细胞功能的关键基因网络，这与NHD的发病机制相关。我们使用GeneClip U1发夹克隆系统（GeneClip U1 Hairpin cloning system, Promega）构建了靶向DAP12序列的siRNA载体（SI）及靶向随机序列的对照载体（SCR）。通过Lipofectamine LTX试剂将上述载体转染至THP-1细胞中，并在添加200 μg/ml潮霉素B（Hygromycin B）的培养培养基中培养约两个月以筛选稳定细胞系。随后采用有限稀释法，以每孔1个细胞的密度将细胞接种于96孔细胞培养板，最终筛选得到2个SI克隆（SI5与SI17）及2个SCR克隆（SCR1与SCR4）。