Research Paper Volume 4, Issue 11 pp 843—853

Figure 4. COL16A1 expression is downregulated in senescent HDFn cells and its 3'-UTR is direct miR-181a target.

(A) Predicted miR-181a target site on human COL16A1 3'UTR was identified by TargetScan 6.2 software. (B) Real Time RT-qPCR was employed to analyze the expression level of COL16A1 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). Col16a1 protein level is shown and β-actin was used as loading control. (D) Insertion of COL16A1 3'UTR target sequence in a luciferase reporter vector leads to diminished luciferase activity in presence of miR-181a 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 COL16A1 in proliferating HDFn cells transfected with miR-181a 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-181a versus a scrambled control sequence (ctr). Itga5 protein levels is shown and β-actin was used as a loading control.