Transcriptomic analysis of ZHX3-depleted HepG2 cells

The aim of this RNA-Seq analysis was to identify target genes of the transcription repressor ZHX3 in HepG2 cells. To do so, four stable lines were generated in HepG2 using three different ZHX3-targeting CRISPR constructs and an empty vector construct. Bulk RNA-Seq analysis was performed on these four lines. Sequencing was done by NovogeneAIT Genomics using the Illumina® NovaSeq6000-PE150 platform and 63-82 million base pairs were sequenced per sample. A total of 47 and 156 genes were found to be consistently upregulated and downregulated (fold change ≥ 2 and FDR < 0.05), respectively, in all three KO lines as compared to the empty control line. Functional enrichment analysis of the 156 upregulated genes using g:profiler revealed the enrichment of two biological processess, urate transport and urate metabolic process. qPCR analysis validated the upregulation of uric acid transporter gene SLC17A1. Four different HepG2 stable lines were generated by transfecting with four different CRISPR constructs: (1) empty vector control (termed as Empty), (2) sgZHX3-2 (Z2) , (3) sgZHX3-5 (Z5) and, (4) sgZHX3-9 (Z9). Each stable line was seeded in three wells of a 24-well plate (triplicates).

本RNA测序（RNA-Seq）分析旨在鉴定HepG2细胞中转录抑制因子ZHX3的靶基因。为实现该研究目标，研究人员利用三种靶向ZHX3的成簇规律间隔短回文重复序列（CRISPR）构建体与一种空载体构建体，在HepG2细胞中构建了四株稳定细胞系。随后对这四株细胞系开展批量RNA测序（Bulk RNA-Seq）分析。测序工作由诺禾致源基因（NovogeneAIT Genomics）完成，采用Illumina® NovaSeq6000-PE150测序平台，每个样本的测序数据量为6300万至8200万碱基对。相较于空载体对照组，在全部三株敲除（KO）细胞系中，分别有47个和156个基因呈现持续上调和下调（差异倍数≥2且错误发现率FDR<0.05）。采用g:Profiler工具对156个上调基因进行功能富集分析，结果显示其显著富集于两个生物学过程：尿酸盐转运与尿酸盐代谢过程。实时定量聚合酶链反应（qPCR）分析验证了尿酸转运蛋白基因SLC17A1的上调表达。研究人员通过转染四种不同的CRISPR构建体，共获得四株HepG2稳定细胞系：(1) 空载体对照组（命名为Empty）；(2) 靶向ZHX3的单导RNA（sgRNA）sgZHX3-2（简称Z2）；(3) 靶向ZHX3的单导RNA（sgRNA）sgZHX3-5（简称Z5）；(4) 靶向ZHX3的单导RNA（sgRNA）sgZHX3-9（简称Z9）。每株稳定细胞系均接种于24孔板的3个孔中，设置三次生物学重复。