Open AccessRNAi Supress VEGF, TERT, and Bcl‐xl in HEp‐2 Cells
Author(s) -
Tao ZeZhang,
Yan Wang
Publication year - 2008
Publication title -
otolaryngology–head and neck surgery
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.232
H-Index - 121
eISSN - 1097-6817
pISSN - 0194-5998
DOI - 10.1016/j.otohns.2008.05.514
Subject(s) - small hairpin rna , rna interference , microbiology and biotechnology , in vivo , plasmid , apoptosis , mtt assay , biology , transfection , viability assay , cell growth , chemistry , cell culture , rna , gene , gene knockdown , biochemistry , genetics
Problem 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 pEGFP‐shVEGF‐shTERT‐shBcl‐xl was constructed. Plasmids containing single shRNA against each target (VEFG, 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. Conclusion 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. Significance Our study provides experimental evidence for the clinical application of this technology in the future. Support This work was supported by the grants from the National Natural Science Foundation of China (No. 30471873, No.30672313 and No.30740012).