However, the restoration of miR-195 expression significantly suppressed the ability of HCC cells to promote HUVEC migration (Fig. 2A and Supporting Fig. 1A). Furthermore, compared with the control media (SFM), TCM from NC-transfected or nontransfected HCC cells promoted the HUVECs to develop more capillary-like structures. Tube formation was reduced dramatically in HUVECs that http://www.selleckchem.com/products/LBH-589.html were grown in TCM

from miR-195 transfectants to a level comparable to that of HUVECs cultured in SFM (Fig. 2B and Supporting Fig. 1B). In contrast, the suppression of endogenous miR-195 in HCC cells resulted in enhanced HUVEC migration and capillary tube formation (Fig. 2C,D). To elucidate the role of miR-195 in HCC metastasis, the effects of miR-195 on the migration and invasion of HCC cells were analyzed initially in vitro. Transwell assays showed that both the migratory and invasive activities of HCC cells were suppressed by miR-195 expression (Fig. 3A,B and Supporting Fig. 2A,B) but were promoted

when cellular miR-195 was neutralized by anti–miR-195 (Fig. 3C,D). To further validate the above findings in vivo, orthotopic liver implantations were conducted with the QGY-miR-195-LUC cell line (Supporting Fig. 3). Mice that were injected with QGY-miR-195-LUC were fed with doxycycline for 10 days before being divided into miR-195–on and miR-195–off groups, followed by doxycycline withdrawal

in the miR-195–on group and continued doxycycline treatment in the miR-195–off Selumetinib concentration mice for another 40 days. The tumor sizes remained similar between the two groups before the 40th day postimplantation but were reduced in the miR-195–on group on the 50th day (Supporting Fig. 4A,B; 上海皓元医药股份有限公司 tumor incidence for miR-195–off versus miR-195–on groups: 6/8 versus 5/8). Furthermore, the miR-195–on group displayed fewer MVD in the xenografts and fewer metastases in the liver and lung compared with the miR-195–off group on the 50th day (Supporting Fig. 4C-E). To exclude the potential confounding effect that decreased angiogenesis and metastasis in the miR-195–on group might result from smaller tumors with fewer metabolic demands and fewer cells, an independent experiment was performed to assess tumor angiogenesis and metastasis on the 40th day, the time point when the tumor incidence (miR-195–off versus miR-195–on groups: 9/15 versus 8/14) and tumor sizes (Supporting Fig. 5) were comparable between the groups. As observed in the results on the 50th day, the miR-195–on group had a much lower MVD (Fig. 4A), decreased metastasis incidence (miR-195–off versus miR-195–on groups: 7/9 versus 4/8), and reduced sizes and numbers of metastatic nodules in the liver (Supporting Fig. 6A,B and Fig. 4B,C) and lung (Supporting Fig. 6C,D and Fig. 4D,E) compared with the miR-195–off group.