Correlation Between PC4and VEGF-C/D-VEGFR-3Signaling Molecules Of Lymphangiogenesis In A Nude Mice Model Of Lung Adenocarcinoma

Background and objective:Tumor lymphatic metastasis is a critical factor to affect the prognosis of malignant tumor patients. The human positive cofactor4(PC4) is a lead factor for tumor occurrence and development including lymphangiogenesis in lung cancer. Vascular endothelial growth factor-C (VEGF-C), VEGF-D and VEGFR-3construct a major signal pathway to participate in tumor cell lymphatic metastasis and lymphangiogenesis. Our early research has suggested that there is a positive correlation between PC4and VEGF-C/D-VEGFR-3in vitro. In this study, we will establish a nude mice model of human pulmonary adenocarcinoma lymphatic metastasis and detect the relationship between PC4and VEGF-C/D-VEGFR-3signal pathway.Methods:Twenty BALB/C nude mice were divided equally into four groups. A549cells were injected subcutaneously into the left back near axillar fossa, right back near axillar fossa, left hind limb and left hind nail pad of nude mice in different groups, respectively. Tumor size and lymphatic metastasis were dynamicly observed to screen the optimal animal model of human transplanted pulmonary adenocarcinoma lymphatic metastasis.Other fifteen BALB/C nude mice were divided equally into three groups. A549cells (group A), A549-siRNA PC4cells (group B)and A549-siRNA control cells (group C) were injected subcutaneously into the left back near axillar fossa in different groups, respectively. Lymphatic metastasis in the three groups were observed. Then we detected the mRNA and protein levels of PC4,VEGF-C, VEGF-D,VEGFR-3, LYVE-1in tumor and metastasis lymph node tissue by real time quantitative PCR, Western blot and immunohistochemistry. Meanwhile, we analysed the correlation between PC4and VEGF-C/D-VEGFR-3by using SPSS13.0statistical analysis software.Results:Transplanted pulmonary adenocarcinoma appeared more early and grew faster in the left and right back near axillar fossa groups than that in the other two groups (P＜0.05). Lymph node metastasis rates were41.7%(5/12),42.9%(6/14),23.1%(3/13) and21.4%(3/14) in the left back near axillar fossa, right back near axillar fossa, left hind limbs and left nail pad groups, respectively. The metastasis rates in the left and right back groups were higher than the other two ones (P＜0.05). Injection into the left back near axillar fossa was more convenient than the right one.Lymphatic metastasis rates were41.7%(5/12),25.0%(3/12) and38.5%(5/13) in group A, B and C, respectively. PC4mRNA and protein levels were reduced obviously in tumor and metastasis lymph node tissue in group B (P＜0.05). Along with the lower PC4expression, VEGF-C, VEGF-D, VEGFR-3protein and mRNA levels were declined. The LYVE-1protein expression was also declined in tumor. Further more, the PC4and VEGF-C, VEGF-D, VEGFR-3protein levels were positively correlated in both tumor (r=0.991,P=0.047; r=0.997, P=0.039; r=0.986,P=0.01) and metastasis lymph node (r=0.982,P=0.012; r=1,P=0.02; r=1, P=0.015).Conclusion:1. Subcutaneous injection of A549cells in the left back near axillar fossa of nude mice has the advantages of convenience, fast tumor growth, and high lymph metastasis rate. It might be an optimal animal model of human transplanted pulmonary adenocarcinoma lymphatic metastasis.2. In the human transplanted pulmonary adenocarcinoma nude mice models, the expression of PC4is positively related with VEGF-C,VEGF-D and VEGFR-3. PC4may be a upstream molecules of VEGF-C/D-VEGFR-3signal pathway and take part in regulating tumor lymphangiogenesis and lymphatic metastasis.