Pevelopment And Primary Application Of A Method For Quantitative Detection KLK4 Gene Expression

The human tissue kallikrein is a subgroup of serine proteinase family wich contains 15 genes. It expresses in many kind of tumor tissue, especially in hormonal related tumor tissue and concerned with clinical stage. KLK4 is one of this family. The expression of KLK4 gene is correlated closely with the growth of ovarian, prostate and other cancers. So it may has potential value of clinical applications.Objective: To develop a real-time quantitative PCR method for detection of human breast cancer related KLK4 gene expression and to investigate KLK4 expression levels in breast cancer and normal tissues.Methods: Using Sybr Green I, with GAPDH as reference, a real-time quantitative PCR method was established. KLK4 expression levels of 64 normal and breast cancer tissues were detected. Performed statistical analysis by clinical pathoindex.Results: The amplification efficiencies for GAPDH and KLK4 of the real-time quantitative PCR method were 0.98 and 0.96. Inter-coefficient of variation were 0.8% and 1.6%, respectively; intra--coefficient of variation were 3.9% and 2.4% respectively. The DNA sequence analysis of gene amplification product and melting curve analysis showed the method wasreliability.The level of KLK4 mRNA in normal breast and breast cancer tissue were 0.0120 + 0.0044 and 0.0272 + 0.0067 (P<0.0\). The level of KLK4 mRNA in breast cancer tissue were as follows: in ER group, ER(+) was 0.0269 + 0.0070 and ER(-) was 0.0276 + 0.0067; in PR group, PR(+) was 0.0270 + 0.0065 and PR(-) was 0.0275 + 0.0081; in CerbB-2 group, CeybB-2(+) was 0.0270 + 0.0064 and CerbB-2(-) was 0.0301+0.0074; in tumor metastasis and no-metastasis group were 0.0258 + 0.0061 and 0.0276 + 0.0066 respectively. There were no significantly different in eachConclusions: This research developed a method for KLK4 mRNA with FQ-PCR. It is convenient, good reproductive and high reliability, offering an ideal detection method of KLK4 gene expression.The level of KLK4 mRNA in breast cancer tissue is higher significantly than that in normal breast tissue. This indicates that the KLK4 gene expression has relevance with breast cancer onset. Its expression level have nothing to do with ER, PR, CrebB-2 and tumor metabasis.