Research Paper Volume 4, Issue 11 pp 843—853

Figure 3. miR-152 represses ITGA5 expression and controls HDFn cell adhesion.

(A) Predicted miR-152 target sites on human ITGA5 3'UTR were identified by TargetScan 6.2 software. (B) Real Time RT-qPCR was employed to analyze the expression level of ITGA5 in HDFn cells at a growing number of passages in culture (p1-p16). Values reported are the average ± SD of three independent experiment. (C) Western blot of HDFn cells protein extracts collected at increasing passage number in culture (p1-p16). Itga5 protein level is shown and β-actin was used as loading control. (D) Insertion of ITGA5 3'UTR target sequence in a luciferase reporter vector leads to diminished luciferase activity in presence of miR-152 in HEK293 cells 24h after co-transfection. Histograms show the values resulting as the average ± SD from three independent co-transfections.(E) Real Time RT-qPCR was employed to analyze the expression level of ITGA5 in proliferating HDFn cells transfected with miR-152 versus a scrambled control sequence (Ctr). Values reported are the average ± SD of three independent experiment. (F) Western blot analysis of protein extracts of HDFn transfected with miR-152 versus a scrambled control sequence (Ctr). miR-152 overexpression decreases ITGA5 protein levels; β-actin was used as a loading control. (G) Adhesion assay performed on proliferating HDFn cells transfetcted with miR-152 versus scrambled control sequence. (H) Histogram shows adhesion ability of proliferating HDFn 96h after transfection with control or miR-152. Values reported are the average ± SD of three independent experiment. *p-Value <0.01 by Student's t test.