Quantitative real-time reverse transcription-PCR assay for urokinase plasminogen activator, plasminogen activator inhibitor type 1, and tissue metalloproteinase inhibitor type 1 gene expressions in primary breast cancer.
BACKGROUND
The plasminogen activation system and matrix metalloproteinases (MMPs) play a key role in the degradation of basement membrane and extracellular matrix in tissue remodeling, cancer cell invasion, and metastasis.


METHODS
Quantitative real-time reverse-transcription-PCR (RT-PCR) assays were developed to quantify urokinase-type plasminogen activator (uPA), plasminogen activator inhibitor type 1 (PAI-1), and tissue metalloproteinase inhibitor type 1 (TIMP-1) mRNA in 54 breast cancer tissues. Gene fragments were amplified in a LightCycler real-time PCR system using gene-specific primers and SYBR Green I. The results were normalized to beta-actin mRNA. We also quantified antigen and functional concentrations of these components.


RESULTS
The intra- and interassay variabilities for mRNA quantification showed mean SDs for the crossing point of 0.12 and 0.15 cycles, respectively. PAI-1, uPA, and TIMP-1 mRNA and antigen concentrations and PAI-1 and uPA functional concentrations increased with tumor severity; the increase was statistically significant for PAI-1, uPA, and TIMP-1 mRNA and antigen concentrations and for uPA functional concentrations. Node-positive patients showed significantly higher PAI-1, uPA, and TIMP-1 mRNA and antigen concentrations than those who were node negative.


CONCLUSIONS
Quantitative real-time RT-PCR is a highly sensitive, reproducible, and fast method for measuring gene expression of PAI-1, uPA, and TIMP-1 in breast cancer. These components may be involved in breast cancer development, and increased mRNA expression may be associated with a worse prognosis.
Castelló
Remedios
R
Hospital Universitario La Fe, Centro de Investigación, Avda. Campanar 21, 46009 Valencia, Spain.
Estellés
Amparo
A
Vázquez
Carlos
C
Falcó
Cristina
C
España
Francisco
F
Almenar
Sergio M
SM
Fuster
Carlos
C
Aznar
Justo
J
D000328:Adult; D000368:Aged; D001940:Breast / Q000378:metabolism; D001943:Breast Neoplasms / Q000378:metabolism* / Q000473:pathology; D005260:Female; D006801:Humans; D008875:Middle Aged; D017395:Plasminogen Activator Inhibitor 1 / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D012720:Severity of Illness Index; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000235:genetics / Q000378:metabolism*; D014568:Urokinase-Type Plasminogen Activator / Q000235:genetics / Q000378:metabolism*
D017395:Plasminogen Activator Inhibitor 1; D012333:RNA, Messenger; D019715:Tissue Inhibitor of Metalloproteinase-1; D014568:Urokinase-Type Plasminogen Activator