Genome-wide gene expression changes in KSRP silenced breast cancer cells


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Raquel Duarte1,Kiashanee Moodley1,Caroline Dickens1,Valcarcel Juan2
1University of the Witwatersrand,2Centre for Genomic Regulation

Abstract

Background

Epithelial- to- mesenchymal transition (EMT) bestows cancer cells with properties that are essential for malignant progression. During EMT epithelial cells undergo cellular changes ultimately assuming a mesenchymal phenotype. The RNA binding protein KSRP has recently been shown to influence TGF-β induced changes in gene expression during EMT. Previous profiling of epithelial vs mesenchymal breast cancer cells have identified KSRP as being differentially expressed in epithelial cells. We decided to investigate the genome –wide changes that accompany KSRP silencing in an epithelial breast cancer cell line.

Method

MDA-MB-468 cells were transfected with siRNA directed to KSRP. Scrambled siRNA was used a control. Gene expression profiles and transcript isoforms were determined using the Affymetrix GeneChip® Array 2.0 at the CRG core facility. Genome-wide changes in KSRP knockdown MDA-MB-468 cells were compared to SiScr MDA-MB-468 cells.

Results

Results of the Affymetrix GeneChip Human Transcriptome array analysis identified genes, transcription factors, splicing factors, exons and splicing patterns altered by silencing KSRP.  49 genes had a fold change ≥ 1.5 (p value ≤ 0.05) were regulated upon KSRP knockdown in MDA-MB-468 cells.  Among this group were three members of the Wnt signaling pathway- CCND3, PPP2R1B and DKK1. Of the 1624 differentially expressed transcription factors 8 had a ≥ 1.5 fold change in expression. 7 differentially regulated exons from 4 distinct genes were identified and 190 differentially regulated alternative splicing patterns from 149 distinct genes were altered. We present the genes from the various categories. Results from the gene chip analysis were validated by qRT-PCR analysis.  

Conclusion

The Wnt signaling pathway is the only pathway regulated in response to KSRP knockdown. The three upregulated genes are all functional in the canonical Wnt pathway and not in the planar polarity or Wnt/Ca2+ pathway. Findings in this study suggest a specific regulatory role for KSRP in canonical Wnt signaling.