Silencing of TRIM28 in human iPSC

TRIM28, a multi-domain protein, is crucial in the development of mouse embryos and the maintenance of embryonic stem cells (ESC) self-renewal potential. As the epigenetic factor modulating the structure of chromatin, TRIM28 regulates the expression of numerous genes and is associated with progression and poor prognosis in many types of cancer. IPSC served as a model of the cells with stem cell-like phenotype, e.g., cancer stem cells. We evaluated the role of TRIM28 in pluripotency maintenance in iPSC by silencing endogenous TRIM28 expression with siRNA (iPSC-siTRIM28). IPSC treated with siRNA with no target sequence served as a control (siCTRL) of the experiment. Cells lacking TRIM28 lose the expression of pluripotency markers, as well as the ability to self-renew, and they start to differentiate. Pathway enrichment analysis using Gene Ontology datasets showed significant upregulation of pathways related to apoptosis, differentiation, cellular response to DNA damage stimulus and cell cycle regulation in iPSC-siTRIM28, relative to reference iPSC (iPSC-WT and iPSC-siCTRL). Analysis was performed on 1 biological replicate, 3 technical replicates, 14 days upon siRNA treatment. Samples were compared to control iPSC (treated with control siRNA, with no target), and to wild-type iPSC (non-treated).

TRIM28是一种多结构域蛋白，在小鼠胚胎发育以及胚胎干细胞（Embryonic Stem Cell, ESC）自我更新潜能的维持中发挥关键作用。作为调控染色质结构的表观遗传因子，TRIM28可调控众多基因的表达，并与多种癌症的进展及不良预后密切相关。诱导多能干细胞（induced pluripotent stem cell, iPSC）可作为具有干细胞样表型细胞的研究模型，例如肿瘤干细胞。我们通过小干扰RNA（small interfering RNA, siRNA）沉默内源性TRIM28的表达（构建iPSC-siTRIM28组），以此评估TRIM28在iPSC多能性维持中的作用。以转染无靶向序列siRNA的iPSC作为实验对照（siCTRL组）。缺失TRIM28的细胞会丧失多能性标志物的表达能力与自我更新潜能，并启动分化程序。基于基因本体（Gene Ontology, GO）数据集进行通路富集分析结果显示，相较于对照iPSC（iPSC-WT组与iPSC-siCTRL组），iPSC-siTRIM28组中与细胞凋亡、细胞分化、DNA损伤应激应答以及细胞周期调控相关的通路显著上调。本次分析仅设置1次生物学重复与3次技术重复，实验于siRNA转染后14天开展。样本分别与对照iPSC（转染无靶向序列的对照siRNA处理组）以及野生型iPSC（未处理组）进行比较。