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In a previous study by the current authors,hepatocellular carcinoma (HCC) was determined to beepidemiologically sex-dependent, and the incidence andmultiplicity of HCC found to decrease in estradiol-3 benzoate(EB)-treated F344 rats. Therefore, to ascertain the anticancermechanism of EB, a commercially available cDNA microarray,with a total of 14,815 cDNA rat gene clones, was used todetermine the differentially expressed genes in nontreated livers,EB-treated livers, and diethynitrosolamine (DEN)-inducedHCC. In the sequenced experiment, a total of 85 genes weredifferentially expressed at either two or more times the rate ofthe normal expression, where 33 genes were downregulatedby EB, and 52 genes upregulated. Candidate genes wereselected according to significant changes observed in themRNA expression in the EB-treated livers compared with thenontreated livers, then these genes were filtered accordingto their different expression patterns in the DEN-inducedtumors compared to the estrogen-treated livers. To confirmthe microarray data, a real-time PCR analysis was performedfor ten selected genes: the H-ras revertant protein 107 (Hrev107),insulin-like growth factor binding protein (IGFBP),parathyroid hormone receptor (PTHR), SH3 domain bindingprotein (SH3BP), metallothionein, src-suppressed C-kinasesubstrate (SSeCK) gene, phosphodiesterase I, CD44, epithelialmembrane protein 3 (EMP3), and estrogen receptor α (ERα).The SSeCK and phosphodiesterase I genes were bothupregulated in the DEN-induced hepatocarcinomas, yet theirpossible carcinogenic functions remain unknown. Meanwhile,the other genes were downregulated, including the genes relatedto growth regulation (IGFBP, H-rev107, ERα), adipogenesisinhibition (PTHR), and tumor suppression (metallothionein).

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