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.