ABSTRACT
Background: Puma is a highly robust pro-apoptotic protein. The protein becomes activated by p53 ensuing beyond-repair DNA damage. Down regulation 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