A del T poly T (8) mutation in the 3' untranslated region (UTR) of the CDK2-AP1 gene is functionally significant causing decreased mRNA stability resulting in decreased CDK2-AP1 expression in human microsatellite unstable (MSI) colorectal cancer (CRC).
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.
Shin
Joongho
J
Department of Surgery and Molecular Genetics, Albert Einstein College of Medicine, New York, NY, USA.
Yuan
Ziqian
Z
Fordyce
Kenneth
K
Sreeramoju
Prashanth
P
Kent
Tara S
TS
Kim
John
J
Wang
Victor
V
Schneyer
David
D
Weber
Thomas K
TK
D020413:3' Untranslated Regions / Q000235:genetics; D045744:Cell Line, Tumor; D015179:Colorectal Neoplasms / Q000235:genetics* / Q000503:physiopathology; D017353:Gene Deletion; D015972:Gene Expression Regulation, Neoplastic; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D053842:Microsatellite Instability; D009690:Nucleic Acid Conformation; D020871:RNA Stability / Q000235:genetics*; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics; D012334:RNA, Neoplasm / Q000737:chemistry / Q000235:genetics; D025521:Tumor Suppressor Proteins / Q000235:genetics*
D020413:3' Untranslated Regions; C096151:CDK2AP1 protein, human; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D025521:Tumor Suppressor Proteins; D049452:Green Fluorescent Proteins