Skipping of exon 10 in<i> Axl</i> pre-mRNA regulated by PTBP1 mediates invasion and metastasis process of liver cancer cells

Abstract

The Axl gene is known to encode for a receptor tyrosine kinase involved in the metastasis process of cancer. In this study, we investigated the underlying molecular mechanism of Axl alternative splicing. <b>Methods</b>: The expression levels of PTBP1 in hepatocellular carcinoma (HCC) tissues were obtained from TCGA samples and cell lines. The effect of <i>Axl-L</i>, <i>Axl-S</i>, and PTBP1 on cell growth, migration, invasion tumor formation, and metastasis of liver cancer cells were measured by cell proliferation, wound-healing, invasion, xenograft tumor formation, and metastasis. Interaction between PTBP1 and Axl was explored using cross-link immunoprecipitation, RNA pull-down assays and RNA immunoprecipitation assays. <b>Results</b>: Knockdown of the PTBP1 and exon 10 skipping isoform of Axl (Axl-S), led to impaired invasion and metastasis in hepatoma cells. Immunoprecipitation results indicated that <i>Axl-S</i> protein binds more robustly with Gas6 ligand than <i>Axl-L</i> (exon 10 including) and is more capable of promoting phosphorylation of ERK and AKT proteins. Furthermore, cross-link immunoprecipitation and RNA-pulldown assays revealed that PTBP1 binds to the polypyrimidine sequence<b>(TCCTCTCTGTCCTTTCTTC)</b> on <i>Axl</i>-Intron 9. MS2-GFP-IP experiments demonstrated that PTBP1 competes with U2AF2 for binding to the aforementioned polypyrimidine sequence, thereby inhibiting alternative splicing and ultimately promoting <i>Axl-S</i> production. <b>Conclusion</b>: Our results highlight the biological significance of <i>Axl-S</i> and <i>PTBP1</i> in tumor metastasis, and show that PTBP1 affects the invasion and metastasis of hepatoma cells by modulating the alternative splicing of <i>Axl</i> exon 10.