Identification by a Digital Gene Expression Displayer (DGED) and test by RT-PCR analysis of new mRNA candidate markers for colorectal cancer in peripheral blood.
Evidence from the literature widely supports the efficacy of screening for colorectal cancer (CRC) in reducing mortality. A blood-based assay, potentially, represents a more accessible early detection tool for the identification of circulating tumour cells originating from a primary tumour site in the body. The present work aimed at identifying a set of specific mRNAs expressed in colon tissue but not in blood cells. These mRNAs may represent useful markers for early detection of circulating colon cancer cells by a simple, qualitative RT-PCR assay, following RNA extraction from peripheral blood samples. Using a data-mining tool called cDNA digital gene expression displayer (DGED), based on serial analysis of gene expression (SAGE) from the Cancer Genome Anatomy Project (CGAP) database, 4-colon and 14-blood cDNA libraries were analyzed. We selected 7 genes expressed in colon tissue but not in blood and were able to test 6 of them by RT-PCR in peripheral blood of CRC patients and healthy controls. We present a relatively easy and highly reproducible technique for the detection of mRNA expression of genes as candidate markers of malignancy in blood samples of patients with colon cancer. SAGE DGED provided a list of the best candidate mRNAs predicted to detect colon cells in the blood, namely those encoding the following proteins: hypothetical protein LOC644844 (LOC644844, whose cDNA was not amplifiable), fatty acid binding protein 1 (FABP1), carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5), mucin 13 cell surface associated (MUC13), guanylate cyclase activator 2A (GUCA2A), amiloride binding protein 1 (ABP1), galactoside-binding, solute carrier family 26, member 3 (SLC26A3). The mRNA expression of these genes was evaluated in 8 samples from subjects diagnosed with CRC and 9 from healthy controls. We observed the expression of 2 of the 6 investigated genes in the blood samples of the vast majority of patients considered, but also in a subset of the controls. Our data confirm the extreme sensitivity of RT-PCR, making this technique able to detect minimal amounts of mRNA expressed in a non-tissue-specific manner. Moreover, DGED remains a powerful tool to identify candidate epithelial markers in blood, such as colon related mRNAs. However, to date, none of these qualified as tumour markers.
Lauriola
Mattia
M
Dipartimento di Istologia, Embriologia e Biologia Applicata, Università di Bologna, I-40126 Bologna, Italy.
Ugolini
Giampaolo
G
Rosati
Giancarlo
G
Zanotti
Simone
S
Montroni
Isacco
I
Manaresi
Alessio
A
Zattoni
Davide
D
Rivetti
Stefano
S
Mattei
Gabriella
G
Coppola
Domenico
D
Strippoli
Pierluigi
P
Taffurelli
Mario
M
Solmi
Rossella
R
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D014408:Biomarkers, Tumor / Q000032:analysis* / Q000235:genetics; D002277:Carcinoma / Q000097:blood / Q000175:diagnosis / Q000235:genetics*; D016022:Case-Control Studies; D015179:Colorectal Neoplasms / Q000097:blood / Q000175:diagnosis / Q000235:genetics*; D005260:Female; D020869:Gene Expression Profiling / Q000295:instrumentation / Q000379:methods*; D056726:Genetic Association Studies; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000379:methods; D008297:Male; D008875:Middle Aged; D009360:Neoplastic Cells, Circulating / Q000737:chemistry / Q000378:metabolism / Q000473:pathology; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012815:Signal Processing, Computer-Assisted
D014408:Biomarkers, Tumor; D012333:RNA, Messenger