Urokinase-type Plasminogen Activator Receptor High And Low Metastatic Human Lung Giant Cell Carcinoma Cell Lines Regulation Of Expression

The urokinase-type plasminogen activator (uPA) receptor (uPAR) is a 55-60KDglycoprotein which is anchored on the cell-surface membrane by aglycosyl-phosphatidylinositol linkage and specifically recognizes pro-uPA and activeuPA by their epidermal growth factor terminal domains. As receptor-bound uPAcatalyses the formation of plasmin on the cell surface to generate the proteolyticcascade that contributes to the breakdown of basement membranes and extracellularmatrix, uPAR is believed to be of vital importance in cancer cell invasion andmetastasis. The increasing data showed that uPAR is over-expressed in a variety ofcancer cell lines. uPAR levels are correlated strongly with metastatic potential inhuman cancer cell lines and the high levels of uPAR may predict a high risk of cancermetastasis. The regulation of expression of the uPAR gene is therefore important forunderstanding normal cell migration and tumour metastasis and may be critical in thecontrol of cancer invasion and metastasis. In this report, we used high-(95D) and low-metastatic(95C) human lung cancercells to study the transcriptional regulation of uPAR expression. Our data presentedhere would provide the important insights into the transcription regulation of theuPAR gene in particular and enhance our understanding of the general moleculargenetic mechanisms applied to a wider range of genes. The analyses with bioinformatics tools indicate that although there is no typicalTATA box and CCAAT box, several potential transcription factor binding sites werefound in the uPAR promoter. To unveil the molecular mechanisms underlyingtranscription controls of the uPAR gene, we use a list of molecular genetic approachesbelow: 1, analyse the correlation between uPAR expression level and invasive abilityby western blot and Matrigel invasion assay; 2, Perform PCR to obtain the uPARpromoter respectively from 95C and 95D strains and analyse the effect of differentbases of uPAR promoter between 95C and 95D cell on promoter activity; 3, findingthe important promoter region responsible for the different expression of uPAR in 95Cand 95D strains by progressive 5'deletion of the full length promoter and transienttransfection/reporter assay; 4, characterization of the cis-elements by 18bp linkerscanning mutant in uPAR promoter; 5, assessing the methylation state of thepromoter CpG of uPAR gene using methylation-specific PCR method inconjunction with DNA sequencing. 6, Electrophoretic Mobility Shift Assay (EMSA)and RT-PCR were used to analyse the expression profile of important transcription 4factors in 95C and 95D strains. Our results showed that: 1, The uPAR protein level in high-metastatic 95D cells was much higher than that in low-metastatic 95C cells, which indicated that uPAR levels are correlated strongly with metastatic potential in human cancer cell lines. 2, According to the results of sequencing, five bases different are found in uPAR promoter between 95C and 95D cells. The results of luciferase activity assay showed that these differences have no significant effect on the uPAR promoter activity. 3, Based on a normal uPAR promoter, progressive truncated mutants were constructed. The transient transfection/reporter assay showed that the promoter region from -136 to +9 relative to the transcription start site may interacts with relevant nuclear factors, which results in different levels of uPAR expression between 95C and 95D cells. 4, Linker scanning mutagenesis and luciferase activity assay showed that two Sp1 binding sites and a NF-κB bingding site may play a important role in uPAR gene transcription. 5, CpG methylation within the uPAR promoter was not correlated with the low activity of uPAR gene promoter in 95C. 6, Exogenous introduced transcription factor Sp1 and NF-κB increased the promoter activity sharply by transient transfection/reporter assay.