Application of combination of short hairpin RNA segments for silencing VEGF, TERT and Bcl-xl expression in laryngeal squamous carcinoma.
OBJECTIVE
To develop an RNA-interference (RNAi) approach involving the hitting of multiple targets by a recombinant plasmid and evaluate its antitumor effect on laryngeal squamous carcinoma in vitro and in vivo.


METHODS
A plasmid containing 3 different short hairpin RNA (shRNA) segments termed pEGFPshVEGF-shTERT-shBcl-xl was constructed. Plasmids containing single shRNA against each target (VEGF, TERT, BCL-xl alone) individually were also constructed as control. Cells were treated with these plasmids. The expression of targeted genes as well as apoptosis of tumor cells were evaluated after treatment with multiple shRNA vectors or control vectors. The mRNA and protein expression were determined by RT-PCR and western blotting. Cell viability was examined using the MTT assay. Apoptotic morphological alterations were observed by Hoechst staining and electron microscopy. The in vivo antitumor effect was characterized in a nude mice model of laryngeal squamous carcinoma.


RESULTS
We demonstrated that a recombinant plasmid containing multiple shRNAs could effectively and simultaneously inhibit VEGF, TERT and Bcl-xl mRNA and protein expression in the HEp-2 cells; the plasmid containing the 3 different shRNAs exhibited a potent antitumor effect on LSCC both in vitro and in vivo, and could much more effectively induced cell apoptosis than each single shRNA. We also demonstrated that the simultaneous blockage of these 3 genes have a better inhibitory effect on human HEp-2 cells than the blockage of each single shRNA.


CONCLUSIONS
Our study demonstrates that the application of vector-based RNAi technology that involves hitting multiple targets will be a promising therapeutic modality in the gene therapy of human laryngeal cancers; furthermore, it provides experimental evidence for the clinical application of this technology in the future.
Wang
Yan
Y
Department of Otolaryngology-Head & Neck Surgery, Renmin Hospital of Wuhan University, Wuhan, China.
Tao
Ze-Zhang
ZZ
Chen
Shi-Ming
SM
Xiao
Bo-Kui
BK
Zhou
Xu-Hong
XH
Liu
Jun-Ping
JP
D000818:Animals; D002294:Carcinoma, Squamous Cell / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D002470:Cell Survival; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007822:Laryngeal Neoplasms / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008819:Mice, Nude; D009368:Neoplasm Transplantation; D009369:Neoplasms / Q000378:metabolism; D034622:RNA Interference; D019098:Telomerase / Q000096:biosynthesis* / Q000235:genetics; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis* / Q000235:genetics; D051020:bcl-X Protein / Q000096:biosynthesis* / Q000235:genetics
C494811:BCL2L1 protein, human; D042461:Vascular Endothelial Growth Factor A; D051020:bcl-X Protein; C509186:TERT protein, human; D019098:Telomerase