The construction and characterization of a novel adenovirus vector of artificial microRNA targeting EGFR.

Objective: To construct a recombinant adenovirus with artificial microRNA targeting the epidermal growth factor receptor (EGFR) to inhibit the proliferation and induce the apoptosis of tumor cells.

Methods: An artificial microRNA (amiR) targeting EGFR was designed and cloned into an adenovirus shuttle plasmid to obtain pDC312-SLPI (secretory leukoprotease inhibitor)-EGFR amiR-pA. Then it was co-transfected into 293 cells with the adenovirus backbone plasmid pBHGloxΔE1, 3Cre to pack the recombinant adenovirus Ad-SLPI-EGFR amiR. After the recombinant adenovirus infected Hep-2 cells by Ad-SLPI-EGFR amiR, a morphological change was observed, and an MTT assay was performed to assess the proliferation of the tumor cells. The expression of EGFR was determined by western blotting analyses to assess the effect of Ad-SLPI-EGFR amiR.

Results: The sequence of artificial microRNA targeting EGFR was confirmed by DNA sequencing. Ad-SLPI-EGFR amiR significantly downregulated EGFR expression at the protein level and exerted an inhibitory effect on proliferation in Hep-2 cells. The transfected cells became round, shrunken, partly grape-like and floating under a lighted microscope.

Conclusion: The recombinant adenovirus of Ad-SLPI-EGFR amiR was successfully constructed, and it was able to sufficiently decrease the expression of EGFR and inhibit the proliferation of Hep-2 cells.