Figure 7: AFP expression is increased in the liver of anti-miR122 transgenic mice.

(a) DNA construct used to establishment transgenic mice in which miR122 is functionally silenced (anti-miR122 transgenic mice). This construct (like the construct used in the in vitro studies) generates a functional single-stranded full-length antisense miRNA complementary to miR122. (b) Confirmation of the expression of an antisense RNA directed against miR122 in anti-miR122 transgenic (anti-miR122 Tg) mice. Amounts of miR122 detected by in situ hybridization (blue/purple staining) in the liver tissues of anti-miR122 transgenic mice and WT mice. Results shown are representative of three independent experiments performed using littermates from four different mouse lines. Scale bar, 100 μm. No specific staining was observed when a negative control probe (LNA-scramble) was used. (c) AFP mRNA expression in mouse liver tissues was analysed by quantitative RT–PCR. Data represent the mean±s.d. of results for five mice in each group. *P<0.05 (t-test). (d) CUX1 and ZBTB20 expression in mouse liver tissues was assessed by Western blotting. A representative result from three independent experiments is shown. (e) Levels of miR214 in liver tissues were determined by quantitative RT–PCR. Data represent the mean±s.d. of three independent experiments. *P<0.05 (t-test). (f) Protocols of the orthotopic xenograft models of HCC cells. Control and miR122-silenced PLC/PRF/5 cells were prepared and injected under the capsule of the left-lobe of the nude mouse liver. Six mice were included in each group. At 4 weeks after transplantation, liver tissues were collected and sliced in series. Histological examination by H&E staining was performed to examine the tumour cell invasion status. (g) Representative liver histology images at 4 weeks after transplantation of tumour cells are shown. Whereas only the transplanted HCC cells beneath the capsule of the liver edge could be detected (arrow, upper left panel), neither intrahepatic metastasis nor vascular invasion was observed in the livers of mice transplanted with control cells. In contrast, vascular invasion by multiple tumour cells (arrow, upper right panel) was observed in mice transplanted with miR122-silenced cells. Scale bar, 500 μm. WT, wild type.