Figure 3.

Overexpression and Inhibition of miRNA199a and Its Effect on the Expression of Reporters Containing miRNA199a Binding Sites at the 3′ UTR

To investigate the effects of inhibiting miRNA199a on the expression of reporter genes with binding site at the 3′ UTR, Hepa1-6 cells were co-transfected with either CMV-EGFP or CMV-EGFP-miR199a*3 and miRNA199a inhibitor (A) and either CMV-GLuc or CMV-GLuc-miR199a*3 and miRNA199a inhibitor (C). The percentage of GFP-positive cells for each group was quantified with flow cytometry and expressed as the percentage of GFP-positive cells after transfection with CMV-EGFP. Similarly, the amount of secreted luciferase was quantified and expressed as a percentage of quantity of secreted luciferase after transfection with CMV-GLuc for each treatment group. (B) Representative fluorescent microscopic and flow-cytometric images for each treatment group. (D) The effects of overexpressing miRNA199a on reporter expression in HCC cell lines by co-transfecting either CMV-GLuc or CMV-GLuc-miR199a*3 and miRNA199a mimic were studied. The secreted luciferase was quantified and represented as the percentage of secreted luciferase after transfection with CMV-GLuc for each treatment group. A two-tailed t test was performed to compare the difference between indicated groups using GraphPad 7.0 (data are reported as mean ± SD, n > 3; *p < 0.05).