Study On The Transcriptional Regulation Of Urokinase Plasminogen Activator In Human Lung Cancer

Metastasis is the main reason that cause the death of most patients with malignancy. Tumor metastasis is an multi-step and complex process which is implicated in the degradation and remodeling of extra-cell matrix. A series of protease such as Fibronolysin, matrix metalloprotease(MMP), cathepsin are involved. Urokinase plasminogen activator (abbreviated as uPA) play an important role in these processes . uPA is an serine protease and is secreted initially as a 54kDa single-chain zymogen of inactive molecule, binding of uPA to its cell surface receptor termed uPAR lead to the high concentration of uPA on the cell surface . the active uPA can hydrolysis the plasminogen enzymatically and convert it into plasmin by its serine protease domain. Degradation of the components of cell matrix directly or activate the MMP promote the invasion and metastasis of tumor cells. Binding of uPA to uPAR can mediate the cell signal transduction which cause the expression of the gene responsible for cell proliferation, cell migration and angiogenesis and facilitate tumor invasion and metastasis. Many clinical research shows that there's an obvious decrease in survival as the expression of uPAR goes up.Although post-transcriptional and translational regulation take part role in the uPA gene regulation, it has been well documented that the gene regulation of uPA is mainly at transcriptional level. So, study on the mechanism of uPA transcriptional regulation is very important to decrease the degradation of extra cell matrix and to depress the tumor metastasis. At present, the uPA promoter has been cloned and sequenced. Studies have shown that an enhancer containing two AP1 sites is located 1980bp upsteam of uPA gene transcriptional start site. The enhancer play an important role in regulation of uPA promoter activity. There is an 74bp cooperation mediator region(abbreviated as COM) between the two AP1 sites. It was reported that the COM region was divided into two parts, uCOM(caatcagcatgacagcct) and dCOM(atctggagtcatgagagct) are located upstream and downstream of this region respectively. There are four transcriptional factors called UEF1,UEF2,UEF3,UEF4 binding to the uCOM region and the UEF1 and UEF2 bind to the dCOM region, cooperation of these four factors binding to the COM region and the transcriptionalfactors binding to the AP1 site function as an complete enhancer to regulate the uPA promoter activity. At present, the UEF2, UEF3 and UEF4 have been identified, but the UEFl which play an important role in both uCOM and dCOM region is still unknown. Characterization and identification of UEFl will facilitate to understand the mechanism of uPA transcriptional regulation.To identify the UEFl which can bind to the dCOM region of uPA enhancer and its effect on the regulation of uPA promoter activity, we construct the cDNA library of 95 C and 95D cell strain and screen the cDNA library by using the dCOM region as a bait sequence in a yeast one-hybrid system in order to identify the protein which can bind to the dCOM region of uPA enhancer. Twenty colonies can grow on the SD/-Leu/-His plate with 45mmol/L 3-AT. Three colonies have the 3 -galactosidase activity in a colony lift assay among them. Only two positive clone can be obtained at last. The two positive clone have the same sequence. After submitted to Genbank and run BLAST programe, the DNA sequence is homology to the cDNA sequence of PBK1 gene. Based on the sequence of PBK1 gene, the full-length of open reading frame of PBK1 cDNA was amplified from the cDNA library and was inserted into the MCS of pET28a(+) proeukaryotic expression vector. The recombinant was identified by sequencing and induced by IPTG The 6xHIS-PBKl fusion protein was purified by Ni-NTA column affinity chromatography and dialysis. Electrophoresis mobility shift assay(EMSA) was performed by using the sequence of dCOM region(atctggagtcatgagagct) labeled 32p as probe. We confirmed that the PBK1 protein can form the DNA-Protein complex with dCOM and the complex has the same mobility with the dCOM-UEFl complex from the 95D cells.PBK1 protein has a domain which is partly homologous to ribosome LI subunit when PBK1 protein sequence is submitted and aligned with data in Genbank. Two putative nuclear localization sequence (NLS) was found at the 3'-terminal of PBK1. To investigate the cellular localization of PBK1, Based on the PBK1 cDNA sequence, we design primers and amplify the full-length ORF of PBK1 from cDNA library. The DNA fragment was inserted into the pEGFP-C3 eukaryotic expression vector after digested with restriction enzyme EcoRI and BamHI. Truncated mutant of PBK1, which the LI homology domain and two NLS was deleted respectively, was generated by PCR method95 D cell was transfected with the plasmid containing full-length PBK1 and truncated mutants respectively, the cellular localization was determined by using...