Figure 2.

Reporter Gene Expression under the Control of miRNA199a Binding Sites

(A) Principle of miRNA-mediated detargeting: inclusion of binding sites of miRNA expressed at high levels in a cell can limit the expression of transgene in that particular cell, while minimal effects are expected in cells that do not express the miRNA. (B) To study the effect of the presence of miRNA199a binding sites at the 3′ UTR of a transgene, miRNA199a-positive cells HepaRG and Hepa1-6 were transfected with CMV-GLuc and CMV-GLuc-miR199a*3; GLuc expression with CMV-GLuc-miR199a*3 was reported as a percentage of CMV-GLuc. Similarly, miRNA199a-negative (C) HCC cell lines and (D) non-HCC cell lines were transfected with CMV-GLuc and CMV-GLuc-miR199a*3, and the amount of secreted GLuc was quantified. (E) Further confirmation of miRNA199a-mediated gene regulation was performed by transfection of Hepa1-6 cells with CMV-EGFP and CMV-EGFP-miR199a*3; 72 hr post-transfection, the percentage of GFP-positive cells was quantified with flow cytometry, and the percentage of GFP-positive cells after transfection with CMV-EGFP-miR199a*3 was normalized with CMV-EGFP. A two-tailed t test was used for statistical analysis to compare the percent expression of CMV-GLuc-miR199a*3 or CMV-EGFP-miR199a*3 with that of CMV-GLuc or CMV-EGFP, respectively, for both HepaRG and Hepa1-6 using GraphPad Prism 7.0 (data are reported as mean ± SD, n > 3; **p < 0.005; ***p < 0.001).