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

Hepatocellular carcinoma (HCC) is one of the most common malignancies in the world. The unclear underlying molecular mechanisms could provide important theoretical basis for prevention and control of HCC. In this study, chromatin immunoprecipitation-sequencing (ChIP-seq) was performed to analyze the binding sites of zinc fingers and homeoboxes 2 (ZHX2) and its genome-wide target genes, and bioinformatics was used to analyze their gene transcription regulation network. Immunohistochemistry detected the ZHX2 expression in HCC, and its association with clinicopathological characteristics of HCC. Results of RT-PCR and western blot showed that the ZHX2 expression in HepG2 cells was obviously lower compared to normal liver cells. ZHX2 could be amplified in ChIP products and ChIP-seq revealed there were 232 genes binding in promoter regions. GO function analysis 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 immunohistochemistry analysis showed the rates of ZHX2 and PTEN expression in paracancerous tissues were both significantly higher than in the 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.