Background: We have previously published results indicating that decreased expression of CDK2-AP1 in MSI human colorectal cancer is associated with deletion mutations in the poly (T) 8 repeat sequence within the 3′-UTR of the CDK2-AP1 gene. In this study, we test the hypothesis that the del T mutation results in decreased CDK2-AP1 expression by causing reduced mRNA stability. Methods: We introduced wild-type and mutant 3′-UTR sequences fused to a green fluorescent protein (GFP) gene separately into human CRC cell lines and quantified the expression of the GFP gene. Native CDK2-AP1 mRNA stability was measured in human CRC cell lines, using an actinomycin D assay and the mRNA structure folding software mfold 3.2. Results: Mutant GFP-3′-UTR samples demonstrated significantly reduced GFP expression compared with wild-type GFP-3′-UTR as measured by both FACS and real-time PCR. Both the actinomycin D assay and mfold software demonstrated significantly reduced mRNA stability for the del T poly (T) 8 transcript compared with the wild type. Conclusions: In summary, these novel results support our hypotheses that the del T poly (T) 8 observed in the 3′-UTR of the CDK2-AP1 gene in human MSI CRC is functionally significant and results in decreased CDK2-AP1 expression. The results also indicate the mechanism of this decreased expression is caused at least in part by decreased mRNA stability.
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