KIF3B is a rno-miR-127 target gene in proximal tubule cells submitted to H/R.

<p>(A) KIF3B mRNA and protein levels were estimated in NRK-52E cells submitted to H/R. mRNA levels (Upper graph) were determined by quantitative PCR using 28s mRNA as housekeeping control. Fold changes were calculated using Normoxia (Nx) as basal condition. Data are presented as mean±s.e.m. of five independent experiments and asterisks indicate statistical significance (P<0.05) when each sample is compared to normoxia. KIF3B protein levels were estimated by western blotting using actin as loading control. Representative blot from five different experiments is shown. (B) Study of KIF3B mRNA and protein levels in NRK-52E cells transfected with pre/Anti-miR-127. KIF3B mRNA was estimated by PCR in cells treated with pre-miR-127 (left panel) and anti-miR-127 (Right panel). Fold change were calculated using Nx Scramble as basal condition. KIF3B protein levels were assessed by western blotting using Actin as loading control. Only key points of H/R protocol exhibiting significant changes are presented. Illustrative blots from five independent experiments are shown. (C) Luciferase assays of KIF3B 3′UTR vectors, named as S1A and S3B, and empty vector (pGL3-control). Luciferase activity percentage is represented, using renilla activity as normalization control. Data are presented as mean±s.e.m. of three independent experiments and asterisks indicate statistical significance between Pre-miR-scramble and Pre-miR-127 transfected cells in each condition (P<0.05). (Nx: Normoxia; CC: Medium change control; Hyp CM: hypoxia in complete medium; Hyp MM: hypoxia in minimum medium; R-1h: 1 Hour Reoxygenation; R-3h: 3 Hours Reoxygenation; R-6h: 6 Hours Reoxygenation; R-24h: 24 Hours Reoxygenation).