AFP promoter/enhancer +2 driven shRNA (against
P2 promoter) decreased
expression. (A) siRNA target region on the c-Myc P2 promoter. (CpG islands within the target site are marked with *). (B) Various AFP promoter/enhancer fusion constructs up to +2 bp relative to the TSS with downstream c-Myc shRNA (C) Time dependent fall in the expression of c-Myc, by AFPEn–Pr + 2 – myc , in HepG2 cells shows maximum suppression after 5 days of shRNA transfection when compared to its scrambled control (p < 0.05 at all-time points). The apparent increase in c-Myc mRNA on the 6th day, when compared with 5th day, was statistically insignificant (p = 0.25). (D) Significant decrease in c-Myc level was observed in HepG2 by AFPEn–Pr + 2 – myc (p < 0.001) and non- specific positive control CMVPr – myc (p = 0.0026). (E) Same trend was observed in Huh7 after 5 days of transfection by the same two constructs (p < 0.001 and p = 0.015). Basal level of c-Myc, in Huh7, was lesser when compared to that of HepG2. (F and G) No decrease in the expression of c-Myc was observed by AFP promoter/enhancer mediated constructs in Chang Liver and CHO cells (p > 0.05 for both) confirming the specificity of the system. However, significant decrease in c-Myc level in Chang Liver and CHO cells was observed only through CMVPr – myc (p < 0.001 for both). (H) Fall in the expression of c-Myc protein through various shRNA constructs in HepG2, Huh7, Chang Liver and CHO cells corroborated with the RNA level.