48 KB164 KB27 KB199220252004Farthmann, Julianeštevilka:2letnik:389 stranistr. 111-119ISSN:1318-2099COBISSID:18166745URN:URN:NBN:SI:doc-7NVFOFIBenAssociation of Radiology and OncologyRadiology and oncology (Ljubljana)biokemijaBreast NeoplasmsdiagnostikaDojka, novotvorbeJajčnik, novotvorbeNeoplasmsNovotvorbeonkologijaOvarian NeoplasmsPolimerazna, verižna reakcijaPolymerase Chain Reactionrak (medicina)Rna SplicingRna, MessengerRNA, prenašalnaRNA, spajanjetumorTumor Cells, CulturedTumorske celične kultureUrokinaseDevelopment of quantitative RT-PCR assays for wild-type urokinase receptor (uPAR-wt) and its splice variant uPAR-del5|The receptor for the serine protease urokinase-type plasminogen activator, uPAR (CD 87), plays an important role in tumor cell invasion and metastasis ofsolid malignant tumors. uPAR is a highly glycosylated, glycan lipid-anchoredmembrane protein, consisting of three homologous domains. Each individual domain is encoded by two exons: DI by exons 2+3, Dll by exons 4+5, and Dlll by exons 6+7. Beside the wild-type (wt) uPAR mRNA, two splice variants either lacking exon 5 (uPAR-del5) or both exons 4 and 5 (uPARdel4/5) have been described. Previously, we studied expression of the mRNA variant uPAR-del4/5 and uPAR mRNA encompassing exons 2, 3, and 4 (i.e. uPAR-wt plus uPAR-del5) applying real-time RT-PCR assays for quantification of the mRNA concentration. In the present paper, we established two additional specific, robust and highly sensitive RT-PCR assays, based on the LightCycler technology, to specifically quantify either uPAR-wt or its splice variant, uPARdel5. Expression of uPAR-wt and uPAR-del5 was analyzed in different human malignant cell lines (ovarian cancer cell lines OVMZ-6 and OVMZ-10; breast cancer cell lines MDA-MB 231, MDA-MB 231 BAG, MDA-MB 435, and aMCF-7; brain tumor cell line LN 18) as well as in a set of 174 breast cancer tissue samples. uPAR-del5 mRNA was found to be expressed very frequently at a rather low level (typically less than 1% of uPAR-wt mRNA). In tumor tissue from breast cancer patients, a statistically significant correlation between uPAR-del5 and uPAR-wt mRNA (r = 0.779; P < 0.001) was observed. There was no association between the expression level of either mRNA and clinical parameters such as nodal status, tumor size and grade. In estrogen receptor negative tumors, a significantly higher uPAR-del5 expression was found (P = 0.023). The two developed quantitative RT-PCR assays described here may aid further analysis of the function and clinical relevance of uPAR-wt and one of its splice variants, uPAR-del5, in maligReceptor serinske proteinaze plazminogenskega aktivatorja urokinaznega tipa, uPAR (CD 87) igra pomembno vlogo v invaziji tumorskih celic in pri metastaziranju čvrstih malignih tumorjev. uPAR je močno glikoziliran membranski protein, vsajen v glikan-lipidne membrane in sestavljen iz treh homolognih domen. Posamezna domena je kodirana z dvema eksonoma: D1 z eksonoma2 + 3, DII z eksonoma 4 + 5 in DIII z eksonoma 6 + 7. Poleg nemutiraneuPAR-wt RNA, sta bili opisani tudi dve izrezovalni različici, ki bodisi nimata eksona 5 (uPAR-de15) bodisi obeh eksonov 4 in 5 (uPAR-de14/5). Predhodno smo z metodo RT-PCR v realnem času za kvantitativno določevanje mRNAkoncentracije proučevali izražanje mRNA različic uPAR-de14/5 in uPAR mRNA,ki vsebujeta eksone 2,3 in 4 (to je nemutirana uPAR-wt in uPAR-de15). V tem prispevku smo dodatno uveljavili dva specifična, robustna in zelo občutljiva testa RTPCR, ki temeljita na tehnologiji Light-Cyler, da bi specifično določevali vsebnosti uPAR-wt in njegovo izrezovalno različico uPAR-de15. Izražanje uPAR-wt in uPAR-de15 je bilo merjeno v različnih človeških malignih celičnih linijah (ovarijskih rakavih celicah OVMZ-6 in OVMZ-10; rakavih celic dojke MDA-MB 231, MDA-MB 231 BAG, MDA-MB 435 in aMCF-7;možganskih celičnih linijah LN18) kakor tudi v zbiru 174 vzorcev tkiva raka dojke. uPAR-del5 mRNA je bila zelo pogosto izražena v razmeroma nizkih vsebnostih (značilno manj kot 1% glede na uPARwt mRNA). V tumorskem tkivu rakavih bolnikov je bila opažena statistično značilna korelacija med uPAR-de15in uPAR-wt mRNA (r=0.779; P > 0.001). Povezave med izražanjem obeh vrst mRNA in kliničnimi parametri, kakor so status bezgavk velikostjo tumorja in gradusa ni bilo. Značilno povišanje uPAR-de15 pa je bilo opaženo v mailgnihtumorjih z negativnim estrogenskim receptorjem. (Izvleček skrajšan pri2000 znakih)TEXTznanstveno časopisjejournalsSlovenian National E-content AggregatorNational and University Library of SloveniaDruštvo radiologije in onkologije