Bacteria-mediated delivery of RNAi effector molecules against viral HPV16-E7 eradicates oral squamous carcinoma cells (OSCC) via apoptosis.

Delivery of RNAi-mediating shRNA molecules for gene silencing via bacteria, i.e. by transkingdom RNAi (tkRNAi) technology, is suggested to be a powerful alternative technique. In this work, the efficiency of bacterial delivery of shRNAs directed against HPV16-E7-specific mRNA to oral squamous carcinoma cells (OSCCs) was evaluated. E. coli were transfected with a plasmid encoding the inv locus and the Hlya gene to enable the bacteria to enter carcinoma cells and to escape from endocytotic vesicles. The bacterial penetration to the target cells was confirmed by DAPI staining. The HPV16-E7 mRNA expression in bacteria-treated OSCCs dropped to 61% of the controls as measured by qRT-PCR. Corresponding inhibition of the HPV16-E7 protein was confirmed by western blotting. The IC of bacteria-treated OSCCs was reduced to more than 75%. Flow cytometry assays showed higher total apoptosis and caspase-3 activation (6.6-fold and 8.4-fold respectively) in OSCCs following exposure to anti-HPV-E7  bacteria compared to anti-GFP bacteria (2-fold and 2.9-fold, respectively). In conclusion, it was demonstrated for the first time that tkRNAi technology is also useful for treatment of squamous carcinoma cells. Anti-HPV16-E7 shRNA-encoding bacteria can efficiently deliver RNAi effectors to OSCCs mediating a strong and specific gene silencing associated with triggering cell death.