Roles and mechanisms of action of HNF‑4α in�the�hepatic differentiation of WB‑F344 cells

Abstract

Hepatocyte nuclear factor 4 α (HNF‑4α) is a nuclear receptor and mediates hepatic genes. WB‑F344 liver epithelial cells can differentiate into hepatocytes. The present study aimed to examine the roles and mechanisms of action of HNF‑4α on the hepatic differentiation of WB‑F344 cells. WB‑F344 cells were divided into a normal cell group (WB‑F344), empty vector group (PLKO), and gene silencing group (PLKO‑SH). The expression levels of HNF‑4α were measured using reverse transcription‑quantitative polymerase chain reaction analysis. Proliferation of the cells was determined using a Cell Counting kit‑8 assay. Based on western blot analysis, the protein levels of α‑fetoprotein (AFP), albumin (ALB) and cytokeratin19 (CK19) were determined. The positive cell rates of the three groups were assessed using periodic acid‑Schiff (PAS) staining. Following construction of an RNA‑sequencing library, differentially expressed genes (DEGs) between the HNF‑4α‑silenced and normal samples were screened using the limma package and enrichment analysis was conducted using the DAVID tool. Protein‑protein interaction (PPI) and microRNA‑targeted regulatory networks were constructed in Cytoscape software. The PLKO‑SH group exhibited a lower mRNA level of HNF‑4α, higher protein level of AFP, lower protein levels of ALB and CK19, increased cell proliferation, and a lower PAS‑positive cell rate. The HNF‑4α‑silenced and normal samples differed in 499DEGs. In the PPI network, matrix metallopeptidase9 (MMP9), early growth response 1 (EGR1), SMAD family member 2 (SMAD2), and RAS‑related C3 botulinum substrate 2 (RAC2) were key nodes. HNF‑4α may promote the differentiation of WB‑F344 cells into hepatocytes by targeting MMP9, EGR1, SMAD2 and RAC2.