Elsevier

Surgery

Volume 142, Issue 2, August 2007, Pages 222-227
Surgery

Society of University Surgeon
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)

https://doi.org/10.1016/j.surg.2007.04.002Get rights and content

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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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.).

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