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Gene expression changes induced by K-ras oncogene in the human pancreatic duct epithelial cell line (H6c7)

ABSTRACT: k-ras oncogene was transduced into the HPV16-E6E7 immortalized normal human pancreatic duct epithelial cell line (H6c7) to generate a stable K-ras oncogene-expressing cell line (H6c7-Kr). The gene expression profile of the H6c7-Kr cells was compared to that of H6c7 cells. We have excluded (subtracted) genes that were deregulated by transduction of the construct used for K-ras oncogene expression. Keywords: parallel sample

ORGANISM(S): Homo sapiens

PROVIDER: GSE1955 | GEO | 2005/01/11

SECONDARY ACCESSION(S): PRJNA90901

REPOSITORIES: GEO

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Project description:A genome-wide survey of transcriptional targets responding to chronic KRAS/MAPK (mitogen-activated protein kinase) pathway activation in rat ovarian surface epithelial cells (ROSE 199) has identified multiple differentially expressed transcriptional regulators including the architectural transcription factor high mobility group AT hook 2 (HMGA2). To elucidate the role of HMGA2 in the transcriptional network affected by oncogenic signaling, we used an integrated approach combining RNAi-mediated silencing, microarray-based expression profiling, computational prediction of transcription factor binding sites in target gene promoters and phenotypic analysis. Knocking-down HMGA2 resulted in the reversion of epithelial-mesenchymal transition, loss of anchorage independence and in a substantial restoration of the gene expression pattern characteristic of non-transformed ROSE cells. Computational prediction of transcription factor binding sites in the promoters of HMGA2-regulated genes and expression profiling revealed a preferential role of activator complex-1 (AP-1) components Fra-1 and JunB in target gene regulation. Forced expression of HMGA2 did not transform normal ovarian epithelial cells, suggesting that HMGA2 up-regulation is not sufficient for inducing the transformed phenotype, but mediates cancer-specific phenotypic traits in cells expressing mutated KRAS and, hence, oncogene addiction. The Ras signaling target array was designed and produced as described (Tchernitsa et al., 2005). Briefly, we selected 121 genes recovered from subtracted cDNA libraries representing sequences differentially expressed in parental and KRAStransformed ROSE 199 cells (Tchernitsa et al., 2004). In addition, we selected genes from subtracted libraries representing sequences differentially expressed in normal 208F fibroblasts and the HRAS-transformed derivative FE-8 (Zuber et al., 2000). Previously described Ras pathway targets and 13 different genes described as housekeeping genes in many different tissues and cell lines were included to permit normalization of hybridization intensities on microarrays. The total number of gene probes is 325 on the array. Unmodified 70-mer DNA oligonucleotides were chosen as probe sets for the array. The oligonucleotide design was performed with software available on the web (http://oligo.lnatools.com/expression/) using the following parameters: option for LNA frequency=0, melting temperature between 74Â°C and 76Â°C, GC content between 45- 55%, absence of strong secondary structures and proximity to 3â end of coding sequence. Oligonucleotides were synthesized at Illumina, Inc (San-Diego, USA) at the 50 nmol scale. Each of the 325 oligonucleotides was spotted four times at a different location on the slides. Thus, the array contains 1,300 features. Overall the study contains six samples. We studied for conditions: First condition is influence of empty plasmid on ROSE199 cells. No additional hybridization replica. Sample 199_199emptyvector Second condition is influence of siEGFP (used as a scramble duplex) on ROSEA25 cell line. As there was no influence of siEGFP on A25cell line and genes differentially expressed between ROSE199 and ROSEA25 were previously described in our several publication here we also used only one hybridization replica. Sample ROSE199_ROSEA25siEGFP. Third condition is influence of HMGA2 expression vector on ROSE199 cells. One dye swap replica was done. Samples: ROSE199_ROSE199HMGA2 and 199HMGA2_199. Forth condition is influence of siHMGA2 duplex on ROSEA25 cell line compared to ROSE199 cell line. One replica with dye swap. Samples ROSE199_ROSEA25siGMGA2 and ROSEA25siHMGA2_ROSE199.

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Gene expression changes induced by K-ras oncogene in the human pancreatic duct epithelial cell line (H6c7)

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