Cloning of Human PRL-3 Gene Promoter and Preliminary Identification of Its Snail Binding Site / 生物化学与生物物理进展

ABSTRACT

PRL-3 is a key gene related to metastasis of colorectal carcinoma. However, it is known little about the possible regulatory mechanisms of PRL-3 gene expression. There were three possible promoter regions predicted by TRED, a promoter prediction software,which were all located in the upstream regions of PRL-3 gene. One of PRL-3 gene candidate promoters was located in the region of about -1kb upstream proximal to 5′ UTR of PRL-3 gene. Many possible transcription factor binding sites such as Snail, n-MYC, ARNT, E74A, NF-kappaB, NRF-2 and AML-1 were predicted in the region by Consite, a promoter analysis web system. Interestingly, a 5′ CACCTG 3′ core sequence and other related sequences of snail binding sites were found in promoter region of PRL-3 genes. Two PRL-3 gene promoters between -699 to 299 nt and between -642 to -383 nt were cloned into pGL3 vector with luciferase report gene. Both of them had promoter activities in four different cell lines including colorectal carcinoma cell lines SW480 and SW620, nasopharyngeal carcinoma cell line CNE2 and human embryo kidney cell line 293A. Interestingly, the luciferase activities of the short DNA fragmentations with Snail binding site′s core sequence 5′ CACCTG 3′ were higher than that of the longer one. PRL-3 promoter obtaining the 5′ CACCTG 3′ core sequence of Snail binding sites, was validated to bind to snail by chromatin immunoprecipitation (CHIP) analysis and electrophoretic mobility shift assay (EMSA) in SW480 cells. The data suggested that Snail was involved in regulation of PRL-3.