Figure 6

DLEU2 reduces miR-30a-5p expression in ccRCC. (a) Putative DLEU2 RNA binding sequence in miR-30a-5p. (b) Expression levels of DLEU2 in HK-2 cell line and four ccRCC cell lines were examined by real-time PCR. Data are presented as mean±S.D. (P<0.001, independent t-test compared to HK-2 cell line). Experiments were performed three times. (c) MiR report constructs that included one wild-type and two mutant DLEU2 binding sequences were co-transfected into 769-P cells infected with miR-control-lentivirus or miR-30a-5p lentivirus. Relative repression of firefly luciferase expression was normalized against a transfection control. Results are presented as mean±S.D. for three independent experiments. (d) Lysates of 769-P cells underwent RIP with the Ago2 antibody. Left panel: Ago2 was detected using IP-western (left panel), and DLEU2 and miR-30a-5p using real-time PCR. RNA levels are presented as fold enrichment of Ago2 relative to IgG immunoprecipitates (right panel). (e) DLEU2 expression correlated with miR-30a-5p expression in ccRCC samples from the First Affiliated Hospital and Cancer Center of Sun Yat-sen University (left panel), and the TCGA data set from starBASE v2.0 (right panel). (f) DLEU2 expression was inversely associated with survival of ccRCC patients from the First Affiliated Hospital and Cancer Center of Sun Yat-sen University (left panel: X-tile plot and middle panel: Kaplan–Meier analysis for survival and COX proportional hazards model for hazard ratio) and the TCGA data set from TANRIC database (right panel: Kaplan–Meier analysis for survival)