Targeting genes and signaling pathways of transcriptional suppressor ZHX2 in hepatocellular carcinoma: a Chromatin Immunoprecipitation-sequencing (ChIP-seq) investigation

Zili Lv, Rongquan He, Menglan Huang, Guoyu Zhao,  Jie Ma,  Gang Chen

Abstract:

Hepatocellular carcinoma (HCC) is one of the most common malignancies in the world. The unclear molecular mechanisms underlying could provide important theoretical basis for the prevention and control of HCC. This study performed chromatin immunoprecipitation-sequencing (ChIP-seq) to analyze the binding sites between zinc fingers and homeoboxes 2 (ZHX2) and its genome-wide target genes, and bioinformatics was used to analyze their gene transcription regulation network. Immunohistochemistry was used to detect the ZHX2 expression in HCC, and its association with the clinicopathological characteristics of HCC. Results of RT-PCR and western blot showed the expression of ZHX2 in HepG2 cells was obviously lower compared with normal liver cells. ZHX2 could be amplified in ChIP products, then ChIP-seq reveals there were 232 genes binding in promoter regions. GO analysis of functions revealed these genes were mainly associated with biological processes (BP), cellular components (CC), and molecular functions (MF). In addition, PTEN was found enriched in certain biological functions in BP analysis. Then, four pathways of these genes based on Kyoto Encyclopedia of Genes and Genomes (KEGG) were found P<0.05. Last analysis of immunohistochemistry showed the rates of ZHX2 expression and PTEN expression in paracancerous tissues both were significantly higher than that in HCC tissues (P=0.042; P<0.001), with negative correlations with AFP values (r=-0.246, P=0.040; r=-0.263, P=0.028). Further, PTEN expression was positively correlated with the differentiation level in HCC tissues (r=0.267, P=0.025). Spearman correlation analysis revealed that the expression profiles of ZHX2 and PTEN were positively correlated in HCC tissues (r=0.258, P=0.031). This study is the first to use ChIP-seq technology to analyze the specific regulatory mechanisms of the transcription suppressor ZHX2 in the context of HCC at the genome level.