Figure 1: MiR-708 acts as ovarian cancer metastasis suppressor.

(a) Heatmap of 21 most differentially regulated miRNAs from miRNA array of SKOV (Right) and SKOV-I6iv (Left). (b,c) Expression of miR-708 in ovarian tumours by FISH analysis of commercial tissue arrays. Samples were subgrouped by tumor stages or whether metastasis occurred. The statistical significance was determined using χ² test (b). The representative images from different stages were shown in c. Scale bars: 20 μM. (d) Real-time RT-PCR analysis of miRNA-708 levels in SKOV-3, TOV-112D, A1847, A2780, and their derived representative invasive lines. (e) Ovarian cancer cells were transfected with precursor miR-708 or scrambled control (s.c.). Twenty-four hours later, cells were subjected to migration (16 h) and invasion (24 h) assays. (f) SKOV-I6iv cells transfected with miR-708, miR-708 together with anti-miR-708 or s.c. were incubated for migration (6 h) and invasion (8 h) assays. (g) Left: histology of metastasized lungs from mice intravenously injected with SKOV-I6iv cells stably expressing miR-708 (n=9; bottom) or vector control (n=6; up). Images show H&E staining. Scale bars: 100 μM. Right: statistical analysis of total metastatic area in the lung. Ten pictures were taken for each mouse. The dots represent the means of individual mice. Student’s t-test (two-tailed) was used to compare two groups. (h) Left: SKOV-I6iv cells with miR-708 expression or vector control were orthotopically injected into the bursa of the mouse ovary. The kinetics of cancer metastasis to the abdomen cavity was monitored by BLI. Representative BLIs are shown at day 28 after implantation. Right: quantification results of abdominal metastases by BLI measurements (Total BLI minus primary tumour BLI). Data are normalized to the mean of vector control group (n=6). Student’s t-test (two-tailed) was used to compare the two groups. (i) Individual abdominal organ metastasis was measured 30 days after orthotopic implantation using real-time RT–PCR. Human-specific GAPDH levels were used to quantify metastatic SKOV-I6iv human cells. Data represent normalized means±s.e.m. (n=10; data were combined from two separate experiments). Data in d–f represent normalized means±s.d. (n=3). Student’s t-test (two-tailed) was used for statistical analysis (*P<0.05; **P<0.01; ***P<0.001).