Society of University SurgeonA 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)
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Materials and methods
Generation of a GFP 3′-UTR CDK2-AP1 fusion and LV-mediated transfer gene system (3′-UTRCDK2-AP1-pRRL.sin.PPT.hPGK.EGFP-LV): A 3-plasmid Lentiviral Vector mediated gene transfer system (LV-pRRL.sin.PPT.hPGK.EGFP) was kindly provided by Dr. Di Renzo (Turin, Italy) and described elsewhere.11 Briefly, the 3 plasmids used were the packaging plasmid, designed to provide the HIV proteins needed to produce the virus particle; the envelope-coding plasmid for pseudotyping the virion with VSV-G; and the
Protein expression of GFP with the mutant (MT) 3′-UTR compared with GFP with the wild-type (WT) 3′-UTR
To determine the functional significance of the del T alteration within the poly (T) 8 repeat of the 3′-UTR of CDK2-AP1, we used 3′-UTR constructs (WT and MT) linked to a GFP expression system. A Lentiviral vector was used as a mediator for gene transfer (LV-pRRL.sin.PPT.hPGK.EGFP). Different concentrations of both WT and MT 3′-UTR-GFP LV plasmid vector were used to obtain maximum transducing units in MSI CRC cell lines, SW48. At maximal frequency of transduction, MT 3′-UTR-GFP cell lines
Discussion
MSI CRC is characterized by alterations in simple repeat sequences distributed throughout the genome as a result of deficient MMR. When the simple repeat sequence is located within the coding region, a deletion or insertion of nucleotide(s) results in a frameshift mutation, which may lead to loss of gene expression. This mechanism has been demonstrated in the loss of key growth regulatory gene expressions in MSI CRC, including growth factor receptors (TGF-βRII), DNA MMR genes (MSH3),
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Supported in part by an American Cancer Society Research Scholar Award (T.K.W.), a Cancer Research Foundation of America Award (Z.Y.), and a Colon Cancer Challenge Research Scholar Award (J.S.).