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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



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



Clinical Chemistry 48(8): 1288-1295



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.

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Accession: 011236018

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PMID: 12142386


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