E-GEOD-8888 - Transcription profiling of human HCT116 clones stably expressing vegf 5 prime UTR reveals a novel tumor promoting function
Submitted on 28 August 2007, released on 16 June 2008, last updated on 2 May 2014
VEGF165 is one of the key regulators of tumor development. Using HCT116 cells transfected with full-length vegf mRNA, full-length vegf mRNA with mutations of H9D and L14E, mutated vegf mRNAs lacking untranslated regions (UTRs), and 5’UTR mutated between nt 591 and nt 746, we found that vegf 5’UTR resided an anti-apoptotic activity against chemotherapy independently of VEGF165. We next established HCT116 clones stably expressing vegf 5’UTR or the mutated vegf 5’UTR. The cells expressing 5’UTR, but not the mutated UTR, showed the drug-resistant phenotype, anchorage-independent growth, and rapid tumor growth when implanted in athymic nude mice. Microarray and real-time PCR showed that the vegf 5’UTR-expressing tumors up-regulated anti-apoptotic genes including mia and down-regulated pro-apoptotic genes including fas, pdcd1, nrg1, and bax. Microarray analysis also revealed specific down-regulation of IFN-inducible genes (43 genes) in the growing tumors. HCT116 cells stably transcribing vegf 5’UTR decreased STAT1 expression and IFN alpha-STAT1 pathway. In addition to 5-fluorouracil treatment, the vegf 5’UTR-expressing tumors did not respond to IFN alpha therapy at all. This novel tumor-promoting function in the vegf 5’UTR may partly explain the insufficiency of the VEGF-VEGFR strategies and suggest a vegf-targeted silencing strategy as a more effective anti-tumor therapy. Experiment Overall Design: We established HCT116 clones stably expressing vegf 5’UTR (vegf 5’-G5) and vegf 5’UTR mutated between nt 591 and nt 746 (vegf 5’ mut-D5) and implanted into the left and the right flanks of the identical mouse, respectively. The generated tumors on day 14 after injection were removed and total RNA was extracted from each tumor using RNeasy mini (Qiagen). After removing contaminated DNA by DNaseI treatment, 10 micro grams each of RNA of vegf 5’-G5-derived tumor from 3 individual mice was mixed and prepared for microarray analysis as sample RNA. The reference RNA of vegf 5’ mut-D5-derived tumor was arranged in the same manner. The quality of both RNA samples was confirmed using an Agilent 2100 Bioanalyzer. Microarray analysis was then performed: briefly, 1 µg of sample RNA (vegf 5’-G5) and reference RNA (vegf 5’ mut-D5) were converted to cDNA with oligo dT conjugated T7 promoter primer and reverse transcriptase. These cDNAs were amplified using T7 RNA polymerase while labeling with either Cy5-CTP or Cy3-CTP (Low RNA Fluorescent Linear Amplification Kit, Agilent Technologies). 750 ng of Cy3 and Cy5-labeled cRNA were mixed and hybridized on an Agilent 22k human 1A array in a hybridization oven at 65oC for 17 h as rotating. Following hybridization and washing of a slide according to manufacture’s protocol, the arrays were scanned using the Agilent DNA Microarray Scanner (G2565BA) and microarray images were analyzed using Agilent Feature Extraction Software (v7.5). The final signal intensity was resulted from subtracting background signal from row spot signal. The gene expression level was presented as ratio of sample intensity against reference intensity.
transcription profiling by array, unknown experiment type