Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
5513405 | Methods | 2017 | 8 Pages |
Abstract
Post-transcriptional regulation of gene expression by RNA binding proteins (RBPs) and non-coding RNAs plays an important role in global gene expression. Many post-transcriptional regulators are misexpressed and misregulated in cancers, resulting in altered programs of protein biosynthesis that can drive tumor progression. While comparative studies of several RBPs and microRNAs expressed in various cancer types have been reported, a model system that can be used to quantify RBP regulation and functional outcomes during the initiation and early stages of tumorigenesis is lacking. It was previously demonstrated that oncogenic transformation of normal human cells can be induced by expressing hTERT, p53DD, cyclin D1, CDK4R24C, C-MYCT58A and H-RASG12V. Here we describe a user-friendly method for generating this genetically defined model of step-wise tumorigenesis beginning with normal donor-derived human cells. This method immortalizes a donor's normal cells in about a week, reducing the chances of senescence. The entire stable system can be established in less than 12Â weeks. We then demonstrate the utility of such a system in elucidating the expression of multiple RBPs at an early step of tumor formation. We identify significant changes in the expression levels of transcripts encoding RBPs prior to transformation, suggesting that our described donor-derived isogenic system can provide insight about post-transcriptional regulation during the earliest stages of tumorigenesis in the context of diverse genetic backgrounds.
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Authors
Laura S. Bisogno, Jack D. Keene,