Published online Oct 28, 2010. doi: 10.3748/wjg.v16.i40.5122
Revised: July 15, 2010
Accepted: July 22, 2010
Published online: October 28, 2010
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.