Brief Article
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World J Gastroenterol. Oct 28, 2010; 16(40): 5122-5129
Published online Oct 28, 2010. doi: 10.3748/wjg.v16.i40.5122
Inhibition of KIT RNAi mediated with adenovirus in gastrointestinal stromal tumor xenograft
Tian-Bao Wang, Wen-Sheng Huang, Wei-Hao Lin, Han-Ping Shi, Wen-Guang Dong
Tian-Bao Wang, Wen-Sheng Huang, Wei-Hao Lin, Han-Ping Shi, Wen-Guang Dong, Department of Surgery, the First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510080, Guangdong Province, China
Author contributions: Wang TB, Huang WS, Lin WH, Shi HP and Dong WG designed the research; Wang TB performed the research; Huang WS offered new reagents and analytic tools; Wang TB, Shi HP and Dong WG analyzed the data; Wang TB wrote the paper.
Supported by Grants from the Chinese Ministry of Education Funds, No. 20070558266 and Guangdong Provincial Funds for Science and Technology, No. 2007B 031515004
Correspondence to: Tian-Bao Wang, Professor, Department of Surgery, the First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510080, Guangdong Province, China. wangtianbao1@163.com.cn
Telephone: +86-20-85562842 Fax: +86-20-87332617
Received: May 26, 2010
Revised: July 15, 2010
Accepted: July 22, 2010
Published online: October 28, 2010
Abstract

AIM: To investigate a therapeutic method for gastrointestinal stromal tumor (GIST) based on KIT RNA interference (RNAi) with AdMax adenovirus.

METHODS: KIT short hairpin RNA (shRNA), whose lateral sides were decorated with restriction endonuclease sequences, was designed. T4 DNA ligase catalyzed the joint of the KIT shRNA and the green fluorescent protein-containing PDC316-EGFP-U6 to form PDC316-EGFP-U6-KIT. Homologous recombination of AdEGFP-U6-KIT was performed with the AdMax system. Heterotopically transplanted GISTs were established in nude mice. AdEGFP-U6-KIT was intratumorally injected. The volume, inhibition ratio of tumor and CD117 expression of GIST graft tumor in nude mice were compared between test and control groups.

RESULTS: The length of KIT shRNA was determined to be about 50bp by agarose electrophoresis. Gene sequencing detected the designed KIT RNAi sequence in PDC316-EGFP-U6-KIT. After transfection with AdEGFP-U6-KIT, 293 cells displayed green fluorescence. The physical and infective titers of AdEGFP-U6-KIT were 5 × 1011 viral particles/mL and 5.67 × 107 plaque forming units/mL, respectively. The mean volume of the grafted tumor was significantly smaller in test mice than in control mice (75.3 ± 22.9 mm3vs 988.6 ± 30.5 mm3, t = -18.132, P < 0.05). The inhibition ratio of the tumors was 59.6% in the test group. CD117 positive expression was evident in two cases (20%) in the test group and 10 cases (100%) in the control group (χ2 = 10.2083, P < 0.005).

CONCLUSION: AdEGFP-U6-KIT is successfully constructed, and KIT RNAi mediated with Admax vector system can effectively inhibit the expression of the KIT gene and the growth of GIST in nude mice.

Keywords: Gastrointestinal stromal tumor; RNA interference; KIT; Adenoviral vector; Nude mice