Exosome-mediated miRNA delivery promotes liver cancer EMT and metastasis.

The deregulation of exosomal microRNAs (miRNAs) plays an important role in the progression of hepatocarcinogenesis. In this study, we highlight exosomes as mediators involved in modulating miRNA profiles in liver cancer cells after induction of the epithelial-mesenchymal transition (EMT) and metastasis. Initially, we induced EMT in a hepatocellular carcinoma cell (HCC) line (Hep3B) by stimulation with transforming growth factor-β (TGF-β) and confirmed by western blot detection of EMT markers such as vimentin and E-cadherin. Exosomes were then isolated from the cells and identified by nanoparticle tracking analysis (NTA). The isolated exosomal particles from unstimulated Hep3B cells (Hep3B exo) or TGF-β-stimulated EMT Hep3B cells (EMT-Hep3B exo) contained higher levels of exosome marker proteins, CD63 and TSG101. After incubation with EMT-Hep3B exo, Hep3B cell proliferation increased. EMT-Hep3B exo promoted the migration and invasion of Hep3B and 7721 cells. High-throughput sequencing of miRNAs and mRNA within the exosomes showed 119 upregulated and 186 downregulated miRNAs and 156 upregulated and 166 downregulated mRNA sequences in the EMT-Hep3B exo compared with the control Hep3B exo. The most differentially expressed miRNAs and target mRNA sequences were validated by RT-qPCR. Based on the known miRNA targets for specific mRNA sequences, we hypothesized that GADD45A was regulated by miR-374a-5p. Inhibition of miR-374a-5p in Hep3B cells resulted in exosomes that inhibited the proliferation, migration, and invasion of HCC cells. These results enhance our understanding of metastatic progression of liver cancer and provide a foundation for the future development of potential biomarkers for diagnosis and prognosis of hepatic cancer. AJTR