Silencing of RNA binding protein, ZFP36L1, promotes epithelial-mesenchymal transition in liver cancer cells by regulating transcription factor ZEB2

Abstract

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 underlying molecular mechanisms have remained unclear. These proteins function by regulating post-transcriptional gene expression upon binding to the AU-rich elements (ARE's) within the 3’untranslated regions (3’UTRs) of specific mRNAs and increasing their mRNA turnover. Here, we tested the role of ZFP36L1 in hepatocellular carcinoma (HCC) cell lines. ZFP36L1 was under-expressed among the three RBPs in a majority of the HCC cell lines. Silencing 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 of epithelial morphology to elongated mesenchymal morphology and increased migration and invasion potential. Conversely, overexpression of ZFP36L1 abolished these changes. RNA-seq analysis of ZFP36L1-depleted HCC cells revealed a significant upregulation of an EMT-inducing transcription factor, ZEB2 (zinc-finger E-box-binding homeobox 2), and enrichment of pathways associated with mesenchymal cell development and differentiation. ZEB2 mRNA contains AREs within its 3’UTR and its stability was increased following ZFP36L1 knockdown. Conversely, ZEB2 was significantly downregulated following ZFP36L1 overexpression and ZEB2 3’UTR was regulated by ZFP36L1 in luciferase reporter assays. These data identify ZEB2 mRNA as a ZFP36L1 target in HCC cells and demonstrate that ZFP36L1 regulates EMT possibly through direct regulation of ZEB2 mRNA. In summary, our results demonstrate that ZFP36L1 suppresses EMT inliver cancer cells by down-regulating the expression of EMT-inducing transcription factor, ZEB2. These data suggest an important role of ZFP36L1 in the development, progression, and metastasis of hepatocellular cancer.