Preliminary Studies Of Human PRL-3 Gene Promoter And Its Regulatory Elements Of Snail

Phosphatase of Regenerating Liver-3 (PRL-3), a small protein tyrosine phosphatase, is considered as an appealing target for cancer therapy due to its role in metastatic progression. The role of PRL-3 in the pathogenesis of liver metastasis of colorectal carcinoma was firstly identified by Serial Analysis of Gene Expression (SAGE) of colorectal carcinoma. PRL-3 is the sole gene which was consistently highly expressed in 18 specimens from liver metastasis of colorectal carcinoma. Moreover, expression of PRL-3 gene is very low in normal mucosa and primary colorectal carcinoma without metastasis. Recent studies also found that PRL-3 could promote proliferation, migration, invasion and adhesion of cancer cells.PRL-3 belongs to a subclass of PTP family (protein tyrosine phosphates), which has 3 members including PRL-1, PRL-2 and PRL-3. Homology of amino acid sequences among three members exceeds 75%, which suggests their functional similarity. In their catalytic domain, PRLs lack Serine-Threonine residues essential for catalysis of the phosphatases. PRLs have only one catalytic domain, characteristic of a CAAX sequence in C-terminus for prenylation. PRLs is located in the cytoplasm when prenylated and in the nucleus when non-prenylated, which suggests PRLs participate as signal molecules in signal pathway network.PRL-3 is also named as PTP4A3, which is located in human's chromosome 8(8q24.3) .According to its location in genome, several genes such as PTK2,GPR20and TSSK5P1 exist in its neighborhood. PRL-3 gene consists of 5 exons and 4 introns,and there are untranslated regions (UTR) existing in both 5' and 3' end of itstranscripts, which may be related to gene regulation of PRL-3.The relationship between PRL-3 and tumor metastasis has been identified. However, there are still many questions to be answered. In order to elucidate the role of PRL-3 in tumor metastasis, it is important to know the regulatory mechanisms of PRL-3 gene expression.Snail is a transcriptional factor involved in epithelial to mesenchymal transition (EMT). Bioinformatic analysis suggests that there are several binding sites of Snail located in the PRL-3 gene promoter. As an important regulatory factor in tumor metastasis, the role of Snail in transcriptional regulation of PRL-3 gene expression is still unknown. We aim to clarify the role of Snail in transcriptional regulation of PRL-3 gene expression.Methods1. Seven PRL-3 gene promoter DNA fragmentations located in the region from -754bp to 455bp of PRL-3 gene transcript were cloned into pGL3 vector with luciferase report gene. Luciferase report gene constructs of PRL-3 gene promoter were transfected into human embryo kidney cell line 293A and colorectal carcinoma cell lines SW480 and SW620 to detect luciferase activities.2. DNA sequence for regulatory elements of Snail in the region from -642bp to -383bp of PRL-3 gene promoter were mutated, cloned into pGL3 vector with luciferase report gene and transfected into human embryo kidney cell line 293A and colorectal carcinoma cell lines SW480 and SW620 to detect luciferase activities.3. The DNA fragmentations with different length in the region located from -642bp to -383bp of PRL-3 gene transcript were cloned into pGL3 vector with luciferase report gene and transfected into human embryo kidney cell line 293A and colorectal carcinoma cell lines SW480 and SW620 to detect luciferase activities.Results1. Seven PRL-3 gene promoter DNA fragmentations located in the region from -754bp to 455bp of PRL-3 gene transcript were cloned into pGL3 vector with luciferase report gene. Each of them had promoter activities in three different cell lines including human embryo kidney cell line 293A, colorectal carcinoma cell lines SW480 and SW620. The promoter activities of the DNA fragmentations located in the region from -642bp to -383bp of PRL-3 gene transcript was higher than the others.2. There are three possible regulatory elements of Snail in the region from -642bp to -383bp of PRL-3 gene promoter. Regulatory elements of Snail in the region from -642bp to -383bp of PRL-3 gene promoter were succefully mutated and cloned into pGL3 vector with luciferase report gene. The promoter activities of the DNA fragmentations located in the region from -642bp to -383bp of PRL-3 gene transcript were deleted after the regulatory elements of Snail in the region from -624bp to -619bp were mutated. The regulatory elements of Snail in the region from -624bp to -619bp of PRL-3 gene promote may be the key element for PRL-3 regulation.3. Five DNA fragmentations of PRL-3 gene promoter located in the region from -642bp to -383bp of PRL-3 gene transcript were cloned into pGL3 vector with luciferase report gene. Four of them except for DNA fragmentations located in the region from -642bp to -573bp of PRL-3 gene promoter had promoter activities in three different cell lines including human embryo kidney cell line 293A, colorectal carcinoma cell lines SW480 and SW620. The promoter activities of the DNA fragmentations located in the region from -642bp to -503bp of PRL-3 gene transcript was highest than the others.Conclusions1. DNA fragmentations located in the region from -642bp to -383bp of PRL-3 gene transcript, the promoter activities of which was the highest one among seven DNA fragmentations of PRL-3 gene promoter, is a key regulatory element for PRL-3 regulation.2. A Snail binding site in the region from -624bp to -619bp of PRL-3 gene promoter is a key regulatory element for PRL-3 regulation.3. DNA fragmentations located in the region from -642bp to -503bp of PRL-3 gene promoter is suggested as the key element of PRL-3 gene promoter.