Fig. 8
AR influences MVD and MLD by regulating VEGF-A and VEGF-C. After the mice were killed, the primary tumor tissues were fixed with 4% formaldehyde, imbedded in paraffin and processed by immunohistochemistry. a Representatives images of IHC staining of CD34 for microvessel density (MVD) and D2-40 for microvessel lymphatic density (MLD) inside or surrounding primary tumors, respectively. Dashed lines mark the borders between tumor nodules and surrounding tissues. Scale bars in 20× and 40× represent 100 and 50 μm, respectively. b Quantitation of angiogenic and lymphangiogenic vessels (MVD vs. MLD) in the various groups (four mice/group). Mean ± SEM of five random views was calculated for every slide and group. c Metastatic possibility was stratified by low-level (~2/3, N = 38) or high-level (~1/3, n = 20) of MVD/MLD (χ 2-test). d Number of metastatic nodules in lung and/or liver was positively correlated with MVD (linear correlation, r = 0.739, p < 0.001, n = 21). e Representative picture of IHC staining of AR, HIF2α, VEGF-A, and VEGF-C in the primary RCC. Overexpression of AR increased VEGF-A expression while suppressed VEGF-C expression, but knockdown of miR-185-5p could reverse the results of AR. Scale bars, 50 μm. f Quantitation of HIF2α, VEGF-A, and VEGF-C expressions detected by immunohistochemistry using Image-Pro Plus software (four mice in every group). g Real-time PCR assays for miR-185-5p expression in various groups. Total RNAs were subjected to real-time PCR assays and the relative expressions were plotted as distribution plots. The experiments were carried out in triplicate. *and #p < 0.05 were considered statistically significant. *AR compared to control and #Sh-miR-185-5p compared to control. NS no significance (t-test for two groups or ANOVA for more than two groups)
