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. 2018 May 4;9(34):23577–23588. doi: 10.18632/oncotarget.25280

Figure 4. miRNA122a based post-transcriptionally controlled suicide gene therapy for HCC.

Figure 4

To explore the possibility of performing a targeted gene directed enzyme prodrug therapy (GDEPT) utilizing cytosine deaminase/5-Fluorocytosine (CD/5-FC) system; we constructed CMV-CD, and CMV-CD-miR122a*3; the later consisting of 3 miRNA122a binding sites at the 3′-UTR of suicide gene CD. miRNA122a positive cell line HuH7 and miRNA122a negative HCC cell lines were transfected with these plasmids and subsequently incubated with the prodrug 5-FC. (A) The percentage proliferation was quantified with MTS assay and proliferation percentage of cells transfected with CMV-CD-miR122a*3 was reported as percentage of those transfected with CMV-CD. (B) Similarly, total cell death after suicide gene therapy with CMV-CD-miR122a*3 was quantified with flow cytometry by annexin/PI co-staining and reported as relative to CMV-CD. (C) Representative flow cytometric plots for each cell type showing percentage of annexin and PI positive cells after suicide gene therapy (D) Representative flow cytometric plot of HuH7 cell line showing annexin positive cells in the control group overlayed with annexin positive cells after transfection with CMV-CD and CMV-CD-miR122a*3. Two-tailed t-test was performed in Graph Pad prism to check for statistical significance of observed differences. (n > 3, **p < 0.005).