OBJEVTIVETo construct a eukaryotic expression vector of short-hairpin RNA (shRNA) targeting human Akt gene and assess the effect of Akt gene silencing on the growth of colon cancer Lovo cells.

METHODSTwo shRNAs targeting human Akt gene were cloned into pENTRTM/U6 plasmid to obtain the entry clones, and the positive clones were verified by sequencing. After recombination of the pENTRTM/U6 entry constructs and Plenti6/Block-iT DEST vector, the positive clones were confirmed by sequencing. Lovo cells were transfected by the entry vector and DEST Vector, and RT-PCR and Western blotting were performed to detect the interference of Akt gene expressions.

RESULTSThe pENTRTM/U6 entry clones carrying Akt shRNA and pLenti6/DEST-pENTRTM/U6-Akt shRNA were successfully constructed. Both of the vectors were transfected into Lovo cells and resulted in obvious knockdown of the mRNA and protein expressions of Akt.

CONCLUSIONThe Akt siRNA expression vector constructed can significantly inhibit Akt gene expression in Lovo cells, which facilitates further studies of Akt function and tumor gene therapy.

Cell Line, Tumor ; Colonic Neoplasms ; pathology ; Genetic Vectors ; genetics ; Humans ; Proto-Oncogene Proteins c-akt ; biosynthesis ; genetics ; RNA Interference ; RNA, Small Interfering ; genetics ; Transfection