MiR-675-Induced Mesenchymal to Epithelial Transition in HCC Is Associated With a Reduction in TWIST1
Whalen Clark*1, Abul Elahi1, Jonathan M. Hernandez1, Jian Wang1, Leigh Ann Humphries1, Bryan C. Fuchs2, Kenneth Tanabe2, David Shibata1
1Gastrointestinal Oncology, Moffitt Cancer Center, Tampa, FL; 2Surgical Oncology, Massachusetts General Hospital, Boston, MA
Introduction: Epithelial-to-mesenchymal transition (EMT) is a process by which cellular subpopulations in primary epithelial tumors acquire a mesenchymal phenotype and the ability to metastasize. Subsequently, the metastases may revert back to the original epithelial phenotype, by mesenchymal to epithelial transition (MET). Certain microRNAs have been shown to induce EMT, although their role in MET remains poorly defined. AFP-secreting hepatocellular carcinomas (HCC’s) are thought to exhibit a more aggressive behavior and also express high levels of the pro-proliferative miR-675. We sought to evaluate the effects of miR-675 manipulation on known EMT markers and inducers in HCC cell lines.Methods: MiR-675 was transfected into the low miR-675-expressing HCC cell line HepG2 and inhibited by anti-miR in the high-expressing Huh-1 cell line. Successful transfections were confirmed by measurement of miR-675 levels using real-time RT-PCR. Western Blot and/or RT-PCR analyses were used to evaluate changes in the expression of known EMT markers (E-cadherin, Vimentin) and inducers (Twist1, Zeb1, Zeb2).Results: MiR-675 transfection into HepG2 cells resulted in an upregulation of E-cadherin and a concomitant reduction in Vimentin, findings consistent with MET. Conversely, silencing of miR-675 in the Huh-1 overexpressing cells resulted in a mesenchymal (EMT) profile with an upregulation of Vimentin. Among the EMT inducers, Twist 1 was downregulated in HepG2 miR-675 transfectants but upregulated in the anti-miR treated Huh-1 cells. There was no impact of miR-675 manipulation on Zeb1 and Zeb2 in either cell line.Conclusion: Despite being associated with increased proliferation, we have shown that miR-675 induces MET with an attenuation of Twist1, suggesting that the processes of tumor growth and metastasis may be regulated by different pathways. MiR-675 may regulate cellular epithelial reprogramming that allows further growth following the completion of the metastatic process.
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