MicroRNAs miR‐125b and miR‐100 suppress metastasis of hepatocellular carcinoma by disrupting the formation of vessels that encapsulate tumour clusters

We have previously shown that vessels that encapsulated tumour cluster (VETC), a prevalent vascular pattern in hepatocellular carcinoma (HCC), facilitates the entry of the whole tumour cluster into the bloodstream in an invasion‐independent manner, and that angiopoietin 2 (Angpt2), the levels of which are increased in HCC cells, is essential for VETC formation. However, the mechanisms underlying VETC formation remains unclear. Herein, we characterized miR‐125b and miR‐100 as novel VETC suppressors by using human HCC specimens, and cell and animal models. We showed that reduced expression of either miR‐125b or miR‐100 in human HCC tissues was significantly associated with the presence of VETC, venous invasion of tumour cells, and the occurrence of endothelium‐coated microemboli. To confirm the role of miR‐125b and miR‐100 in VETC formation and HCC metastasis, cell lines with stable miR‐125b and miR‐100 expression were established by using human VETC‐2 cells and mouse Hepa1‐6 cells, the hepatoma cells that developed xenografts with VETC patterns. Our results showed that expression of miR‐125b or miR‐100 in VETC‐2 and Hepa1‐6 cells dramatically reduced VETC formation in xenografts, and consequently inhibited in vivo metastasis, suggesting that miR‐125b and miR‐100 may attenuate metastasis by repressing VETC formation. Further investigation revealed that miR‐125b directly suppressed the expression of Angpt2 by binding to its 3′‐untranslated region, whereas miR‐100 reduced the protein level of Angpt2 by targeting mechanistic target of rapamycin (MTOR) and blocking the MTOR–p70S6K signalling pathway. Moreover, the suppressive effect of miR‐125b and miR‐100 on VETC formation was abrogated by injecting Angpt2‐expressing viruses into xenografts. Taken together, our findings imply that miR‐125b and miR‐100 negatively regulate Angpt2 expression through different mechanisms, in turn inhibit VETC formation, and consequently abrogate the VETC‐dependent metastasis of hepatoma cells. This study uncovers new regulatory mechanisms of VETC formation, identifies novel functions of miR‐125b and miR‐100, and provides new targets for antimetastasis therapy of HCC. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.