Transcriptomic analysis of ZHX3-depleted HepG2 cells. 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. Overall design: 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。为达成该研究目标，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）敲除载体与空载体，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NovogeneAIT Genomics采用Illumina® NovaSeq6000-PE150平台完成，每个样本的测序碱基量为6300万至8200万对。与空载体对照细胞系相比，三株敲除（KO）细胞系中共有47个基因持续上调、156个基因持续下调，所有差异基因均满足倍数变化≥2且错误发现率（False Discovery Rate, FDR）<0.05的筛选标准。使用g:profiler对156个上调基因进行功能富集分析后发现，其显著富集于两个生物学过程：尿酸转运与尿酸代谢过程。实时定量聚合酶链式反应（qPCR）验证了尿酸转运基因SLC17A1的上调表达。整体实验设计：通过转染四种不同的CRISPR载体，构建了四株不同的HepG2稳定细胞系：(1) 空载体对照组（命名为Empty）、(2) sgZHX3-2（记为Z2）、(3) sgZHX3-5（记为Z5）以及(4) sgZHX3-9（记为Z9）。将每株稳定细胞系接种于24孔板的三个孔中，设置三次生物学重复。