[SiRNA expression cassettes targeting VEGF effectively inhibit the growth of laryngeal squamous cell carcinoma cell lines].
OBJECTIVE
To study the effects of siRNA expression cassettes (SECs) targeting VEGF in vitro on cultured Hep-2 cells, the observation of the expression of VEGF, the screening of the best interference sequences, and the exploration of the application of RNA interference on tumor gene therapy in the future.


METHODS
On the basis of the principle of target sequence of siRNA, four interference sequences of VEGF were designed, and the downstream gene-specific primers of the SECs were synthesized. The RNAi transcription kit used U6 RNA-based polymerase III promoter and modified terminator for high level, precise siRNA expression inside target cells. The Hep-2 cells was transfected in growth period of index with the PCR products of the four sites separately, and began to observe and measure the results of RNA interference in 48 hours.


RESULTS
The transfected 1366-site cells created, turned into the round shape and began to shed off, whereas morphology of the cells of other groups had not obviously changed. Performing the agarose gels electrophoresis with RT-PCR products, compared with the contrast groups, some cells VEGF mRNA of 1366-site were suppressed obviously, the ratio of OD was 0. 05 while the expression of VEGF of the cell of other groups had not obviously changed. Western blot revealed that VEGF expression was decreased obviously post transfection using 1366-site SECs. Flow cytometry showed that apoptosis rate of 1366-site transfected cells is 43%, and apoptosis rate of the rest three site transfected cells scarcely changed. Similar results were obtained in three independent experiments.


CONCLUSIONS
The study suggested that siRNA expression cassettes (SECs) targeting VEGF 1366-site can effectively inhibit the growth laryngeal squamous cell carcinoma cell lines (Hep-2).
Chen
Niong
N
Department of Otolaryngology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China.
Kong
Wei-jia
WJ
Dong
Ji-hua
JH
Zhang
Dan
D
Zhang
Su-lin
SL
Han
Yue-chen
YC
D017209:Apoptosis; D002294:Carcinoma, Squamous Cell / Q000235:genetics* / Q000473:pathology; D045744:Cell Line, Tumor; D020868:Gene Silencing; D006801:Humans; D007822:Laryngeal Neoplasms / Q000235:genetics* / Q000473:pathology; D034741:RNA, Small Interfering; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics*
D034741:RNA, Small Interfering; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A