Figure 6

miR-206 inhibits in vitro angiogenesis and in vivo tumor growth, angiogenesis and lymphangiogenesis in PDAC xenografts. (a) in vivo tumor growth after re-expression of miR-206 was monitored in PDAC xenograft mouse models. PANC10.05 cells stably overexpressing miR-206 (pBABE-miR-206) or the control vector (pBABE-empty) were injected subcutaneously into the back along the mid-dorsal line of SCID mice (n=7 in case of pBABE-empty and n=8 in case of pBABE-miR-206 tumors). Tumor volume was measured every 2–3 days for 43 days after inoculation. Stable overexpression of miR-206 in PANC10.05 cells was validated by qRT-PCR. Established tumors from PANC-1 cells that were injected s.c. into the back along the mid-dorsal line of SCID mice were randomized into two groups when tumor size reached a size of 0.2 cm³ and were subsequently transfected in vivo weekly with a mixture of miR-206 mimics (n=6) or miRNA-negative control (n=5) in 0.015% collagenase II solution intratumorally. The arrows indicate each intratumoral miRNA injection. Tumor volumes were measured for 17 days after the first intratumoral miRNA injection. All mice were killed 7 days after the last injection. In vivo miRNA transfection efficiency was determined by quantifying miR-206 levels in dissected PANC-1 tumors by qRT-PCR. Data are shown as mean±s.e.m. (b) HUVEC cell proliferation in the presence of conditioned medium derived from PANC-1 cells previously transfected with siRNAs against either ANXA2 or KRAS, as well as with miR-206 mimics or negative controls (n=6/group) was determined after 72 h by MTT assay. Data are shown as mean±s.e.m. (c) HUVEC cell trans-well migration towards conditioned medium derived from PANC-1 cell previously transfected with miRNA mimics, siRNAs or controls (n=6/group) was evaluated by light microscopy. Cells were counted and data are shown as mean number of counted cells/field ±s.e.m. Scale bar, 250 μm. (d) Capillary-like tube formation was determined using HUVEC cells treated with conditioned medium of PANC-1 cells previously transfected with either siRNAs or miRNA mimics for 6 h (n=6/group). Data are shown as mean number of tubes/field ±s.e.m. Scale bar, 250 μm. (e) Whole-mount staining of PANC-1 and PANC10.05 tumors overexpressing miR-206 or negative control for CD31 and LYVE-1 stainings. Scale bar for PANC10.05 and PANC-1 tumors is 50 μm. The quantification of angiogenesis and lymphangiogenesis was determined by calculating the mean of the total area of CD31 and LYVE-1 positive area (μm2) per field ±s.e.m., respectively (n=6–9/group). Data were analyzed by a two-tailed unpaired t-test; *P<0.05, **P<0.01, ***P<0.001.