Short hairpin RNA system to inhibit human p16 in squamous cell carcinoma.
OBJECTIVE
To develop short hairpin RNA (shRNA) vectors and virions to trigger RNA interference against human p16.


METHODS
A modular vector-based shRNA system was used to construct multiple distinct shRNA vectors against the unique exon 1alphaof p16. Target sequences were designed using rigid criteria for length and GC content, along with basic local alignment search tool (BLAST) evaluation to ensure targeting specificity. Individual shRNA-p16 cassettes were then cotransfected with a p16 expression vector and evaluated by Western blot. Cassettes showing a high level of p16 repression were used to construct shRNA-p16 expressing adenoviral and retroviral vectors and tested in a human head and neck squamous cell carcinoma line expressing endogenous and exogenous p16.


RESULTS
Adenoviral and retroviral transfer of shRNA-p16 significantly reduced p16 protein levels, while control constructs left p16 expression unchanged.


CONCLUSIONS
Although p16 is a common target of inactivation in head and neck cancer, its biological role remains ill defined. RNA interference is rapidly becoming the standard to target selected genes of interest for inactivation. We have successfully inhibited p16 expression using shRNA cassettes with strong activity against human p16 and integrated these constructs into adenoviral and retroviral vectors for transient and integrated expression in human cells. Application of this novel modular system to primary human cells will allow the biological consequences of p16 loss to be examined both in vitro and in vivo.
Shin
Jennifer J
JJ
Harvard Program in Otolaryngology-Head and Neck Surgery, Massachusetts Eye and Ear Infirmary, Boston, USA.
Katayama
Takafumi
T
Michaud
William A
WA
Rocco
James W
JW
D000256:Adenoviridae / Q000235:genetics; D000957:Antigens, Viral, Tumor; D015153:Blotting, Western; D002294:Carcinoma, Squamous Cell / Q000235:genetics*; D020868:Gene Silencing; D019942:Genes, p16; D005822:Genetic Vectors; D006258:Head and Neck Neoplasms / Q000235:genetics*; D006801:Humans; D007822:Laryngeal Neoplasms / Q000235:genetics*; D010957:Plasmids; D034622:RNA Interference; D012190:Retroviridae / Q000235:genetics; D014162:Transfection; D014407:Tumor Cells, Cultured
D000957:Antigens, Viral, Tumor