Inhibition of KIT RNAi mediated with adenovirus in gastrointestinal stromal tumor xenograft

查看全文 作　　者：Tian-Bao [1]Wang; Wen-Sheng [1]Huang; Wei-Hao [1]Lin; HanPing [1]Shi; Wen-Guang [1]Dong 机构地区：[1]Department of Surgery the First Affiliated Hospital Sun Yat-sen University Guangzhou 510080 ,Guangdong Province ,China 出　　处：《World Journal of Gastroenterology》索引2010年第16卷第40期,共8页 基　　金：Supported by Grants from the Chinese Ministry of Education Funds, No. 20070558266;Guangdong Provincial Funds for Science and Technology, No. 2007B 031515004 摘　　要：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. T 4 DNA ligase catalyzed the joint of the KIT shRNA and the green fluorescent protein-containing PDC316-EGFP-U6 to form PDC316EGFP-U6-KIT. Homologous recombination of AdEGFPU6-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 se-quencing detected the designed KIT RNAi sequence in PDC316-EGFP-U6-KIT. After transfection with AdEGFPU6-KIT, 293 cells displayed green fluorescence. The physical and infective titers of AdEGFP-U6-KIT were 5 × 10 11 viral particles/mL and 5.67 × 10 7 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 mm 3 vs 988.6 ± 30.5 mm 3 , 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 (χ 2 = 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. 关 键 词：Gastrointestinal stromal tumor RNA interference KIT Adenoviral vector Nude mice