| PTEN was identified as a tumor suppressor gene in 1997 located on human chromosome 10q23.3, which was mutated or deleted in a variety of sporadic cancers. PTEN encodes a dual-specificity phosphatase, which is able to dephosphorylate either phospho-tyrosine or phospho-serine/threonine residues. PTEN, also acts as a lipid phosphatase, specifically cleaves the D3 phosphate of phosphatidylinositol (3,4,5)-triphosphate (PIP3), an important intracellular second messenger, produced by the activity of PI3-kinase. By blocking the activation of Akt, FAK or MAPK pathway, PTEN regulates cellular processes such as proliferation, adhesion, migration, cell cycling, and apoptosis. PTEN implicated not only in tumorigenesis and progression but also in normal embryogenesis development. So it is very important to study the PTEN transcriptional regulation in human hepatocellular carcinoma cells (HHCC).To investgate the phosphotase fouction of PTEN protein, we transfected the wild-type PTEN or mutant PTEN, which lost lipid phosphotase activity or lost both lipid phosphotase activity and protein phosphotase activity, expression plasmids into the BEL-7404 cells, It was found that PTEN can suppress the proliferation, migration, clony formation and cell cycle of BEL-7404 cells, and these effects were mainly attributed to its phosphatase activity. Northern blot and Western blot analysis showed that the PTEN protein level and PTEN mRNA level were decreased in human hepatocellular carcinoma (HHCC) cell lines compared with that in L02 cells. The profile of PTEN protein level in each of 8 cell lines closely parallelized with its PTEN mRNA, which means the variation of PTEN protein mostly dependent on change of PTEN mRNA. The DNA sequencing of PTEN ORF which obtained by RT-PCR showed that there was no any mutation in it. These results demonstrated that the decrease of PTEN expression in HHCC cell lines was not caused by PTEN mutation, and may be to due to the transcriptional or post- transcriptional regulation of PTEN gene.It is well known that the regulation of gene promoter activity plays an important role in gene transcription regualtion. In an attempt to analyze the activity of PTEN promoter in HHCC, we isolated a DNA fragment containing 5'-flanking region and the 5'-untranslated region(5'-UTR)from PTEN gene, and performed series promoter deletion. The deletion analysis of PTEN promoter showed that the fragment of 612bp (-1389/-778) can produce maximum promoter activity in 8 cell lines and the core region of PTEN promoter was resided within the 341bp (-1118/-778) fragment. After the transfection of pGL3-612bp (-1389/-778) into 8 cell lines, we found the profile of PTEN promoter activity was almost parallelized with the profiles of PTEN mRNA and PTEN protein in 8 cell lines. Taken together, we concluded that the downregulation of PTEN expression in 7 HCC cell lines probably does not owing to the mutation of PTEN but maybe mainly attributed to the activity lost of PTEN promoter.To identify the functional cis-elements in the core region of PTEN promoter, 10 potential elements (binding sites) were examined by using 8 bp substitutive mutation respectively in SMMC-7721 cells and L02 cells. It was suggested that two MAZ binding sites, located at the region from -1035 to -1016, are very important for the optimal PTEN promoter activity. Electrophoretic mobility shift assays (EMSA) showed that the MAZ and Sp1 could cross bind to MAZ and Sp1 binding sites. The results of co-transfection of either Sp1 or MAZ expression plasmids with luciferase reporter plasmids contain wild-type or mutated PTEN promoters revealed that both Sp1 and MAZ can induce the activation of PTEN promoter through -1035 to -1016 bases containing MAZ binding sites, but not through the sequence from -937 to -923 containing Sp1 binding site. The results in this study suggested that the zinc-finger proteins Sp1 and MAZ could up-regulate the PTEN expression via binding to the sequence from -1035 to -1016 in SMMC-7721 cells and L02 cells.
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