Project description:We analyzed the effects of the expression of HPV 16 E6 oncoprotein variants (AA-a, AA-c, E-A176/G350, E-C188/G350, E-G350), and the E- Prototype in global gene expression profiles in an in vitro model. E6 gene was cloned into an expression vector fused to GFP and was transfected in C33-A cells. Affymetrix GeneChip Human Transcriptome Array 2.0 platform was used to analyze the expression of over 245,000 coding transcripts. We found that HPV16 E6 variants altered the expression of 431 genes in comparison with EPrototype. The altered genes are involved in cellular processes related to the development of cervical carcinoma, such as adhesion, angiogenesis, apoptosis, differentiation, cell cycle, proliferation, transcription and protein translation. Our results show that polymorphic changes in HPV16 E6 natural variants are sufficient to alter the overall gene expression profile in C33-A cells, explaining in part the observed differences in oncogenic potential of HPV16 variants. We analyzed RNA from 16 cell cultures from the cell line C33-A, with the following experimental conditions: C33-A Non-transfected (WT) and C33-A transfected with: 1) mock, 2) HPV16 E-prototype and 3) HPV16 variants: E-G350, E-A176/G350, E-C188/G350, AAa and AAc, performed by duplicate (biological replicates processed independently N=8x2); using the Affymetrix Gene Chip Human Transcriptome Array (HTA) 2.0 platform. Array signal intensities were analyzed with the Affymetrix expression console and normalized data were analyzed by Affymetrix Transcriptome Analysis Console (TAC).
Project description:C33-A is a Homo sapiens cervix carcinoma cell line. In this experiment we determine the level of gene expression under exponentially growing conditions. The final goal of the experiment is to correlate other epigenetic characteristics from other experiments with gene expression levels. C33-A cells from three independent exponentially growing cultures were recovered and processed for RNA extraction and hybridization on Affymetrix microarrays
