E-GEOD-11865 - shRNA profiling of human Colo16 cells knocked down for Downstreams of hnRNP A2
Submitted on 24 June 2008, released on 13 January 2009, last updated on 1 May 2014
Limited knowledge of the downstream targets of hnRNP A2/B1 has, however, precluded a clear understanding of their roles in cancer cell growth. To define the pathways in which this protein acts we have now carried out microarray experiments with total RNA from Colo16 epithelial cells transfected with an shRNA that markedly suppresses hnRNP A2/B1 expression. The microarray data identified 123 genes, among 22283 human gene probe sets, with altered expression levels in hnRNP A2/B1-depleted cells. Ontological analysis showed that many of these downstream targets are involved in regulation of the cell cycle and cell proliferation and that this group of proteins is significantly over-represented amongst the affected proteins. The changes detected in the microarray experiments were confirmed by real-time PCR for a subset of proliferation-related genes. Immunoprecipitation-RT-PCR demonstrated that hnRNP A2/B1 formed complexes with the transcripts of many of the verified downstream genes, suggesting that hnRNP A2/B1 contributes to the regulation of these genes. Experiment Overall Design: Western blotting was performed to confirm the efficient suppression of hnRNP A2/B1 by shRNA, which was endogenously generated from a pSUPER vector system. A vector carrying a target sequence of firefly was used as a control. The specificity of the hnRNP A2/B1 targeting vector has been established in our previous study [Yaowu He, Melissa A. Brown, Joseph A. Rothnagel, Nicholas A. Saunders, and Ross Smith. Roles of heterogeneous nuclear ribonucleoprotein A/B in cell proliferation. Journal of Cell Science. 2005.118(14):3173-83] Experiment Overall Design: For microarray hybridization, the GeneChip Human Genome U133A platform (Affymetrix) used in this study was printed with 22,283 oligonucleotide probe sets representing 18,400 transcripts and variants, including 14,500 known genes. For a probe set, there were typically 8 probe pairs, each containing a perfect match and a mismatch oligonucleotide. Two chips were prepared for each treatment, allowing 4 comparisons between the hnRNP A2-suppressed and the control cells (samples were not paired).
RNAi profiling by array, unknown experiment type