Bioinformatic Analysis And Preliminary Studies Of Human PRL-3 Gene Promoter

Colorectal carcinoma is a common malignant tumor, which leads to death of alot of people. With development of the national economy and improvement ofpeople's living standards, a high incidence of colorectal carcinomas is consistentlyobserved in some populations with highly caloric food rich in animal fat in ourcountry. Metastasis is the main cause leading to most death of patients with colorectalcarcinoma. Some researches showed that micrometastasis can be detected in morethan a half of patients before radical surgery for colorectal cancer, which usuallyresults in metastasis and recurrence of colorectal carcinoma post-surgery. It is criticalto study molecular mechanisms of metastasis of colorectal carcinoma.It is well-known that PRL-3 is a key gene related to metastasis of colorectalcarcinoma. The role of PRL-3 in the pathogenesis of liver metastasis of colorectalcarcinoma was firstly identified by analyzing gene expression profiles of colorectalcarcinoma based on use of Serial Analysis of Gene Expression (SAGE). PRL-3 is thesole gene which was consistently highly expressed in 18 specimens from livermetastasis of colorectal carcinoma. Moreover, expression of PRL-3 gene is very low in normal mucosa and primary colorectal carcinoma without metastasis. The resultshave been validated by several other groups.PRL-3 is also named as PTP4A3, which is located in human 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 untranslated regions (UTR) exist in both 5' and 3' end of its transcripts, whichmay 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, such as the signal pathwaysPRL-3 participating in, the substrate catalyzed, and the possible regulatorymechanisms of gene expression of PRL-3. In order to elucidate the role of PRL-3 intumor metastasis, it is important to know the regulatory mechanisms of PRL-3 geneexpression.Methods1) PRL-3 promoter and its possible binding sites of transcription factors werepredicted and analyzed by bioinformatical methods. DNA sequence of HumanPRL-3 gene and region within 5kb upstream of PRL-3 gene transcript wasobtained from database of the National Center for Biotechnology Information(NCBI, http://www.ncbi.nlm.nih.gov/mapview). The transcriptional start site(TSS) of PRL-3 was predicted and the nucleotide sequence of-700bp to 300bparound the TSS was found by Transcriptional Regulatory Element Database(http://rulai.cshl.edu/cgi-bin/TRED). The location of relevant sequences ingenome and the distance to PRL-3 transcript were analyzed in genomic databaseof NCBI. The possible binding sites of transcription factors in PRL-3 genepromoter were also predicted by Consite, a promoter analysis web system. 2) Two PRL-3 gene promoter DNA fragmentations were cloned into pGL3 vectorwith luciferase report gene. The DNA fragmentations are located in the regionfrom -699 bp to 299 bp and from -642bp to -383bp of PRL-3 gene transcript, a5'-CACCTG-3'core sequence of Snail binding oligonucleotide sequence wasfound in the latter DNA region. Luciferase report gene construct of PRL-3 genepromoter was transfected into several kinds of cells, such as colorectal carcinomacell lines SW480 and SW620, nasopharyngeal carcinoma cell line CNE2 andhuman embryo kidney cell line 293A, Luciferase activities were detected.3) The chromatin immunoprecipitation and PCR methods, Electrophoretic MobilityShift Assay (EMSA) were performed by using antibody specific for snail toverify bindings of transcription factor snail to the PRL-3 promoter.4) The possible role of Snail in the pathogenesis and progression of colorectalcarcinoma was studied. The expression of snail in SW480 and SW620 cells wasdetected by immunocytochemistry and immunofluorescence technique, and theprotein expression of PRL-3 was also measured by immunohistochemicaltechnique in samples from patients of colorectal carcinoma in different stages,protein expression of PRL-3 in normal mucosa of colon and colonic adenomawere used as controls.Results1) There were three possible promoter regions predicted by TRED, a promoterprediction software. They were all located in the upstream regions of PRL-3 genetranscript. The promoter of a gene is usually located in the region of-3kb and-5kb upstream of gene transcript, especially in the region of about -1kb upstream.Fortunately, the third candidate of PRL-3 gene promoter was located in theregion of about -1kb upstream of gene transcript, which was 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 byConsite, a promoter analysis web system. Interestingly, a 5'-CACCTG-3'coresequence and other related sequences of snail binding sites were found inpromoter region of PRL-3 genes by Consite software.2) Two PRL-3 gene promoter DNA fragmentations located in the region from -699bp to 299 bp and from -642bp to -383bp of PRL-3 gene transcript were clonedinto pGL3 vector with luciferase report gene. Both of them had promoteractivities in four different cell lines including colorectal carcinoma cell linesSW480 and SW620, nasopharyngeal carcinoma cell line CNE2 and humanembryo kidney cell line 293A. Interestingly, the promoter activities of the shortDNA fragmentations with Snail binding sites's core sequence 5'-CACCTG-3'was higher than that of the longer one.3) Two regions of PRL-3 promoter, including the DNA fragmentation obtainingthe 5'-CACCTG-3'core sequence of Snail binding sites, were validated to bind tosnail by chromatin immunoprecipitation (CHIP) analysis of SW480 cells. Thisresult was also proved by electrophoretic mobility shift assay (EMSA).4) The Snail protein was mainly located in cell nucleus of SW480 and SW620.Protein expressions of PRL-3 in groups from colorectal carcinomas, normalmucosa, adenoma and metastatic lymph nodes were significantly different (X~2=92.852, P=0.000). Protein expressions of PRL-3 in adenoma was significantlyhigher than that in normal mucosa (Z=-2.902, P=0.004), and protein expressionsof PRL-3 in metastatic lymph nodes was significantly higher than that incolorectal carcinomas (Z=-4.951, P=0.000), moreover, protein expressions ofPRL-3 in colorectal carcinoma was significantly higher than that in adenoma (Z =-3.572, P=0.000)~-. There were correlation between the expression of Snail andprogression of colorectal carcinoma including metastasis (Z=-2.043, P=0.041)Conclusions(1) PRL-3 gene promoter is located in the region from -699 bp to 299 bp of PRL-3gene transcript.(2) There are several Snail binding sites in PRL-3 gene promoter. PRL-3 gene can beregulated by snail by binding to the promoter of PRL-3 gene in vivo.(3) There is correlation between the expression of Snail and progression of colorectalcarcinoma including metastasis.