Loss of RNA Binding Protein, ZFP36L1, promotes EMT in hepatocellular cancer cells by regulating EMT‐inducing transcription factor ZEB2

Abstract

Introduction RNA binding proteins (RBPs) of the zinc finger protein 36 family, including zinc finger protein 36 like 1 (ZFP36L1) are implicated in cancer, however, the mechanisms have remained unclear. These proteins function by regulating post-transcriptional gene expression by binding to AU-rich elements (ARE's) within the 3’untranslated regions (3’UTRs) of specific mRNA and increasing their mRNA turnover. Here, we tested the role of ZFP36L1 in hepatocellular cancer (HCC) using HCC cancer cell lines. Methods ZFP36L1 was either knocked down in HCC cell lines using siRNA or overexpressed using expression plasmids. The effect of ablation or overexpression of ZFP36L1 was assessed on cellular morphology, cellular migration, and transcriptomic changes through assessment of changes in cellular-shape, wound healing, and RNA-Sequencing. The expression of the three RBPs was determined in various HCC cell lines through quantitative PCR and immunoblotting. Results ZFP36L1 was the most highly expressed among the three RBPs in a majority of the HCC cell lines tested, both at the mRNA and the protein level. Knockdown of ZFP36L1 in two of the seven HCC cell lines resulted in epithelial-mesenchymal transition (EMT) like morphological changes which were characterized by the transition to elongated mesenchymal morphology, significant downregulation of E-cadherin, an epithelial marker, and increased migration potential of these cells in wound healing assays. Conversely, overexpression of ZFP36L1 prevented the morphological changes and the cell migration. Transcriptomic analysis revealed a significant upregulation of EMT-inducing transcription factor, ZEB2 (zinc-finger E-box-binding homeobox 2), following knockdown of ZFP36L1. ZEB2 mRNA contains AREs within its 3’UTR indicating ZEB2 mRNA as a potential ZFP36L1 target in these cells. Conclusions Our results demonstrate that ZFP36L1 suppresses EMT in hepatic cancer cells by down-regulating the expression of EMT-inducing transcription factor, ZEB2. The morphological changes and EMT processes suggest an important role of ZFP36L1 in liver fibrosis and the development of hepatocellular cancer.