Association between the Urokinase Plasminogen Activation System Polymorphisms and NSCLC in Chinese Population

Aim: To evaluate the influence of uPA and uPAR gene polymorphisms in the susceptibility to NSCLC. Methods: A total of 500 NSCLC patients and 500 healthy controls were recruited and matched according to age and gender. The SNPs distributed in uPA and uPAR gene were selected for genotyping. The association between genotype and NSCLC risk was evaluated by computing the OR and 95% CI with multivariate unconditional logistic regression analyses. Results: Patients with the uPAR rs344781 T allele had a reduced risk of developing SCC patients (OR = 0.742; 95% CI = 0.579 0.950; P = 0.0176), especially a risk of developing male SCC patients (OR = 0.722; 95% CI = 0.546 0.954; P = 0.0219). And the uPAR rs344781 C/C allele homozygote was associated with an increased risk of SCC in patients (OR = 1.713; 95% CI = 1.145 2.563; P = 0.0083). However, neither allele frequencies nor genotype frequencies in uPA rs4065 were associated with NSCLC (P > 0.05). Conclusions: Our results revealed that genetic polymorphisms of uPAR rs344781 were associated with the risk of developing SCC (squamous cell carcinoma) in a Chinese population.


Introduction
Extracellular Matrix (ECM) degradation mediated by the urokinase plasminogen activation (uPA) system is an important mechanism involved in both physiological and pathological tissue remodeling [1,2] . Many experimental and clinical studies have demonstrated the association of uPA system activity with cancer invasion and metastasis, including lung cancer [3,4] . Members of uPA system, including uPA and its receptor (uPAR), play critical roles in proteolysis, migration, invasion, and metastasis [5,6] . The median levels of uPA and uPAR expression are higher in lung tumor tissues than the adjacent lung parenchyma.
Lung cancer is the leading cause of cancer death around the world, due to its high mortality and morbidity [7] . In China alone, the incidence and mortality associated with lung cancer

Statistical analysis
All statistical analyses were performed using SPSS 13.0 for Windows (SPSS Inc., Chicago, IL). Hardy-Weinberg equilibrium (HWE) was carried out for all SNPs, cases and controls were compared using the χ2 test with a P-value of < 0.001 being considered statistically significant. The χ2 test was used to assess the frequencies of the selected allele and genotype between the cases and controls. The association between SNPs and NSCLC risk was analyzed by computing the odds ratio (OR) and 95% confidence interval (CI) from multivariate unconditional logistic regression analysis. A two-sided P < 0.05 was considered statistically significant.

Results
500 patients (350 males and 150 females) and 500 healthy controls (259 males and 240 females, and gender information for one control subject was missed) were of Chinese Han origin. For NSCLC patients, 331 had adenocarcinoma (ADC), and 169 had squamous-cell carcinomas (SCC); 280 male and 21 female patients were smokers or former smokers, while 189 male and 14 female of the controls were smokers or former smokers. The studied population were within HWE (P = 0.17109 for uPA rs4065; P = 0.14444 for uPAR rs344781, respectively).

Urokinase Plasminogen Activation System Polymorphisms and NSCLC
SCC patients and controls (P = 0.0263). When analyzing the association between genotypes and the risk of SCC, logistic regression analysis revealed that the uPAR rs344781 C/C allele homozygote was associated with an increased risk of SCC in patients (OR = 1.713; 95% CI = 1.145 -2.563; P = 0.0083). (Table 4,5,6).

Discussion
In the current study, we investigated the association between SNPs (uPA rs4065 and uPAR rs344781) and the risk of developing NSCLC. Indeed, we found that patients with the uPAR rs344781 T allele had a reduced risk of developing SCC patients, especially a reduced risk of developing male SCC patients. And the uPAR rs344781 C/C allele homozygote was associated with an increased risk of SCC in patients. However, neither allele frequencies nor genotype frequencies in uPA rs4065 were associated with NSCLC.
Tumor invasion and metastasis require proteolytic enzymes that destroy the extracellular matrix and enhance the formation of novel blood vessels [11] . The uPA system is thought to play a role in several different processes important to tumor progression including tissue remodeling, chemotaxis, tumor invasion, dissemination, proliferation, and angiogenesis [12,13] . The binding of uPA to uPAR increases the efficiency of plasminogen activation and also serves to localize these proteolytic cascades to the migrating or invading edge of cells [5,14,15] . Binding of uPA and vitronectin also promotes cell adhesion and cell migration. In addition, uPAR also interacts with various cell surface receptors such as integrins, growth factor receptors and endocytic receptor 180 [16][17][18] . These interactions activate diverse signaling pathways including FAK, Src, MAPK and PI3K, leading to EMT, cell proliferation, cell migration and the expression of pro-cancer genes [5,19,20] . These signaling pathways play import-

Urokinase Plasminogen Activation System Polymorphisms and NSCLC
ant roles in NSCLC [21][22][23][24] . In accordance with these studies, our study indicated uPAR maybe susceptibility gene involved in NS-CLC development. In this study, the uPAR rs344781 polymorphism was inversely associated with the development of lung SCC in a Chinese population. C/C homozygote individuals had a 1.713-fold increased risk in developing NSCLC compared to T/T homozygote or C/T heterozygote individuals. In addition we found that T allele had a 0.742-fold reduced risk of developing SCC patients, and a 0.722-fold reduced risk of developing male SCC patients.
Lung cancer is a complex disease resulting from environmental factors, genetic factors, and their interactions [25,26] . The possibility that the relationship between the uPA/uPAR genotypes with NSCLC susceptibility in the study is an ethnic-dependent observation cannot be entirely excluded because multiple risk factors and etiology contribute to the pathophysiology of NSCLC development. Our results are not in agreement with those found in a study by Chuen-Ming Shih [10] . Together with earlier studies [27][28][29] , these differences may primarily be attributed to the different living environments.

Conclusion
In conclusion, our results revealed that genetic polymorphisms of uPAR rs344781 were associated with the susceptibility NSCLC. The results of this study uncover the significant relationship between genetic polymorphisms of uPAR with the susceptibility of NSCLC. However, there are three limitaions in our study. First, the small sample size may be a limitation of the present study. Second, we found that uPAR rs344781 polymorphisms were associated with the development of SCC in a Chinese population, would be better illustrated the effect of uPAR rs344781 polymorphisms in SCC cell proliferation if we preformed functional studies of uPAR rs344781 polymorphisms in vitro. Third, population stratification may have led to a bias.