Synergistic Effect of Expressed miR-128 and Puma protein on Targeted Induction of Tumor Cell Apoptosis

Abstract

Background: Puma is a highly robust pro-apoptotic protein. The protein becomes activated by p53 ensuing beyond-repair DNA damage. Downregulation of SIRT 1, by miR-128, elevates activated p53 that foment Puma indirectly. Objectives: In the present study, we used two-expression Adeno-Associated Virus (AAV) system for co-expression of miR-128 and Puma in order to evaluate apoptotic response; both in the tumor and normal cells, respectively. Materials and Methods: Three recombinant AAVs constructs were generated. The First rAAV bearing Puma under the control of hTERT (p-AAV), the second construct designed such that to carry miR-128 downstream of CMV (mi-AAV), and the last construct comprises of the both CMV-miR-128 and hTERT- Puma. Real-Time PCR and western blotting were used to evaluate expression levels of the transduced genes. Results: MTT assay and DAPI staining shown suicidal effect of each recombinant AAV vectors. p-AAV cytotoxicity was recorded for 62% of the tumor cells, while for normal cells it was only 20% cytotoxic. The second construct, mi-AAV, was not as potent and selective as p-AAV. This construct was shown to be 27% and 16% cytotoxic for BT-474 and HEK-293 cells, respectively. Co-expression of Puma and miR-128 (p-mi-AAV) was accomplished with a selective cytotoxicity toward BT-474. This construct was 85% toxic for tumor cells, although it was only 25% toxic for the normal cell line (HEK-293). Conclusions: In this study, we have shown that not only Puma is able to instigate apoptotic response but also its co-expression along with miR-128 could significantly enhance apoptosis in a synergistic manner.