Screening and identification of down-regulated genes in colorectal carcinoma by subtractive hybridization: A method to identify putative tumor suppressor genes

Objective To search for new putative tumor suppressor genes in colorectal carcinoma. Methods Subtractive hybridization technologies were applied to screen and select genes, the expression of which was down-regulated in colorectal carcinoma. mRNAs uniquely expressed in normal cells but not in colorectal carcinoma were recovered as cDNA (sub-cDNA) after two rounds of subtractive hybridization with mRNA prepared from colorectal carcinoma. The sub-cDNAs were then used as probes to screen a normal human colon cDNA library constructed in lambda-Zap II phage. The DNAs of positive clones were in vivo excised, and partial DNA sequences were analyzed and compared with DNA sequence database Genbank. Results A total of 46 different clones with an average of about 1 kilobases in transcript size was recovered. Among these 46 down-regulated genes in colorectal carcinoma were genes encoding immunoglobulin (n = 32), 40-kDa keratin intermediate filamentous protein or IFP (n = 1), major histocompatibility complex-related protein (n = 1), unrelated structural proteins (n = 10) and gene products yet to be identified (n = 2). RNA dotblot hybridizations confirmed that all 46 clones contained genes that were down regulated and have not been reported before in colorectal carcinoma. Conclusions The results of this study suggested that the 46 clones were down-regulated in colorectal carcinoma, they should be further studied as new putative tumor suppressor genes and could be used as new tumor markers of colorectal carcinoma.