Figure 1.
RUNX3 protein expression in tumor adjacent normal prostate tissues and prostate cancer tissues.
A Representative immunohistochemical photographs were taken at different magnifications in tumor adjacent normal prostate tissue and prostate cancer tissues (Top panel ×100, bottom panel ×400). B Compared with that in the tumor adjacent normal prostate tissue, the overall expression level of RUNX3 in the prostate cancer tissues was significantly lower (P<0.01, χ2 test). C Decreased RUNX3 expression was correlated with TNM stage (P<0.01, χ2 test, comparing I-II versus III–IV).
Table 1.
Patients characteristics and RUNX3 expression.
Figure 2.
Reduction of RUNX3 on the abilities of metastasis in vitro.
A and B Twenty-four hours after transfection, the expression of RUNX3 in PC3 and DU145 cells was evaluated by Western blot. β-actin was used as an internal control. C and D Cell migration assay. Representative fields of migration cells on the membrane (magnifications, ×200). Average migration cell number per field. E and F Matrigel cell invasion assays. Representative images show the cells that invaded through the Matrigel when transfected with RUNX3 plasmid or control. Representative histograph of invaded tumor cells is displayed and number of invaded tumor cells quantified. * indicates significant difference from the controls (*P<0.05, ANOVA).
Figure 3.
Target genes regulated by RUNX3.
A The activity of MMP-2 and MMP-9 were evaluated by Gelatin zymography. B Western blot analysis of the relative protein levels of MMP-2, MMP-9, TIMP-1, TIMP-2 and β-actin in RUNX3 re-expression and control group for both PC3 and DU145 cell lines. C Western blot for the protein expression of MMP-2, TIMP-2 and β-actin in RUNX3 slicing and control group for RWPE-1 cell line. D Western blot analysis for MMP-2 and β-actin expression after knock down of TIMP-2. E Real time PCR for MMP-2 mRNA expression after knock down of TIMP-2. Data are shown as mean ± S.D. *P<0.05.
Figure 4.
Inhibition of angiogenesis induced by RUNX3 expression.
A and B CCK-8 cell proliferation assay was performed to detect the HUVECs proliferation. C and D Representative pictures were taken in situ for tube formation in the supernatant of PC3 and DU145 cells transduced with pFlag-control and pFlag-RUNX3. All experiments were carried out in triplicate. E ELISA for the secretion of VEGF in PC3 and DU145 cells transduced with pFlag-control and pFlag-RUNX3. Data are shown as mean ± S.D. *P<0.05.
Figure 5.
The up-regulation of RUNX3 repressed prostate cancer metastasis in vivo.
A Mice were injected with were injected via tail vein with DU145 cells transfected with the indicated expression plasmids. Groups contained 8 mice. Eight weeks later, mice were sacrificed and the lung metastasis of DU145 cells was measured by macroscopic after autopsy. Histologic analysis of metastatic lesions in the ribs of nude mice was carried by HE staining. B Quantification of nodules in lung with altered RUNX3 levels. The numbers of nodules were blindly evaluated in 5 random fields per implant at 200 magnifications. Data are shown as mean ± S.D. *P<0.05. C Representative immunohistochemical photographs of PAP expression in lung tissue (magnifications, ×200 and ×400). *Tumor tissue.
Figure 6.
RUNX3 expression and angiogenesis in human prostate cancer growing in nude mice.
A Nude mice were injected subcutaneously with DU145 cells stably expressing LV5-RUNX3 or LV5-Control. Western blot was used to examine RUNX3 protein level. B The tumors were monitored for total of eight weeks. Tumor diameter was measured with a caliper every week, and tumor volume was calculated following the formula of A×B2×0.52, where A is the longest diameter of tumor and B is the shortest diameter. The tumor volume was significantly greater at 8 week in mice given control lentiviruses compared with those given RUNX3 lentiviruses (*P<0.05). C Representative mice bearing tumors treated with control compared group with RUNX3 group. D The expression levels of RUNX3 and VIII were analyzed in tumor tissues by immunohistochemistry with representative images showed (magnifications, ×400). E Quantification of microvessel formation in tumors with altered RUNX3 levels. MVD was assessed via vessel counting. The numbers of stained microvessels were blindly evaluated in 5 random fields per implant at 200 × magnifications. * indicates significant difference from the controls (P<0.05).