Restoration of miR-195 in HCC cells significantly suppressed tumor angiogenesis and metastasis in vitro and in vivo. We further provided evidence that miR-195 Romidepsin purchase exerted its antiangiogenic and antimetastatic effects by directly targeting multiple genes, including vascular endothelial growth factor (VEGF), VAV2, and CDC42.

Our findings highlight the importance of miR-195 dysfunction in promoting HCC progression and recurrence and implicate miR-195 as a potential therapeutic target for HCC. Human HCC tissues were collected from 135 patients who underwent HCC resection at the Cancer Center of Sun Yat-sen University between 2001 and 2006. The patients had not received any local or systemic anticancer treatments prior to the surgery, and no postoperative anticancer therapies were administered prior to relapse. All patients were followed postoperatively to assess survival rates and to monitor for recurrence and metastases. The median follow-up time was 45 months. The relevant characteristics

of the studied subjects are shown in Supporting Table 1. Informed consent was obtained from each patient, and the study was approved by the Institute Research Ethics Committee at the Cancer Center. All miRNA mimic and small interfering RNA duplexes (Supporting Table 2) were purchased from GenePharma (Shanghai, People’s Republic of China). Cabozantinib nmr si-VEGF, si-VAV2, and si-CDC42 targeted the mRNAs that coded for human VEGF (NM_001171626.1), VAV2 (NM_003371), and CDC42 (NM_044472), respectively. The negative control RNA duplex (NC) for both miRNA mimic and small interfering RNA was nonhomologous to any human genome sequence. The sequence-specific miR-195 inhibitor (anti–miR-195) and its control (anti-NC) were from Dharmacon (Chicago, IL). The expression vectors pRetroX-miR-195, pMir-vec-LUC, pc3-Gab-VEGF, pc3-Gab-VAV2, and pc3-Gab-CDC42 and firefly luciferase reporter plasmids pGL3cm-VEGF-3′UTR-Wt, pGL3cm-VAV2-3′UTR-Wt, pGL3cm-CDC42-3′UTR-Wt,

pGL3cm-VEGF-3′UTR-Mut, pGL3cm-VAV2-3′UTR-Mut, and pGL3cm-CDC42-3′UTR-Mut were constructed as described in the Supporting Materials and Methods. Human HCC (QGY-7703, MHCC-97H, MHCC-97L, Huh-7, and SMMC-7721), colorectal carcinoma (HCT-116), and transformed human embryonic kidney (HEK293T) cell lines were maintained in Dulbecco’s modified Eagle’s medium (Invitrogen Corp., 上海皓元 Buffalo, NY) that was supplemented with 10% fetal bovine serum (FBS; Hyclone, Logan, UT). The QGY-7703 cell subline, which stably expressed miR-195 and firefly luciferase (QGY-miR-195-LUC), and the matched control line (QGY-control-LUC) were established with the Tet-off system (Clontech, Palo Alto, CA, USA), as described in Supporting Materials and Methods. Human umbilical vein endothelial cells (HUVECs) were isolated and cultured in serum-free medium for endothelial cells (SFM; Invitrogen) as described.[6] HUVECs at passages 2-7 were used.