The Expression Of Endothelial Protein C Receptor And Its Significance In Lung Cancer

Background and objective: Lung cancer is one of the most common malignant tumors, often without symptom at the early stage, easy to miss diagnosis, easy metastasis and recurrence and bad prognosis. There is now an upward trend in the incidence of lung cancer in China year by year. As a common disease, it ranks first in all cause of death of malignant tumors in the world. More and more evidence have shown the linkage between the abnormalities of hemostasis -coagulation and fibrinolysis and most carcinomas. It attracts much attention in the medicine research. Endothelial protein C receptor(EPCR), a new component of the protein C pathway, is a 46-kDa type 1 transmembrane glycoprotein, the EPCR gene is located on chromosome 20q11.2. High expression of EPCR in a variety of tumor cell lines and tumor tissues was found. Many studies indicate activated protein C (APC) -induced effects of cell proliferation, migration and inhibition of apoptosis is related to EPCR. The aim of the present study is to construct small hairpin RNA eukaryotic expression vector specific to EPCR and to observe RNA interference against EPCR effect on tumor cells proliferation, migration and cell cycle in vitro and investigate whether the inhibition of EPCR might lead to slow transplantation tumor and increase host survival using a murine lung carcinoma model.Methods: With endothelial cell as positive control, expression of EPCR on 6 kinds of lung cancer cell and other malignant tumor cell lines and tumor tissues was measured by semi-quantitative RT-PCR, FCM, western blotting and immunohistochemical method respectively. RNAi vector that can express shRNA targeting EPCR(EPCR pshRNA1, 2, 3 vector) and negative control (control shRNA vector) was designed, constructed and transiently transfected into H1299 cells line. Fluorescent real time RT-PCR and Western blotting were employed to detect the change of expression of EPCR mRNA and its protein in H1299. Stably transfected H1299 shRNA-EPCR (H1299/shRNA) and H1299 shRNA-control(H1299/con) cells were obtained and routinely maintained with selection media containing 500μg/mL of G418-sulfate. EPCRmRNA was detected by RT-PCR, and EPCR protein were detected by Western Blot and FCM. MTT method and wound-healing model and FCM analysis were applied to measure cell growth and migration and cell cycle. Some correlated gene expression were also detected. In invivo experiments of tumor growth, H1299/shRNA and H1299/Con and parent cell were injected subcutaneously into SCID murine respectively to construct lung carcinoma xenograft models. Neoplasia formation and growth velocity and life span of murine and microvascular density (MVD) in tumors were evaluated.Results: (1)High expression of EPCR on 6 kinds of lung cancer and other tumorcells as well as cancer tissues were observed. There was a significant difference in thepositive expression rates between tumor tissues(68.3%) and peri-tumor tissues(32.4%).EPCR expression was related to lymphnode metastasis and clinical staging of patients(P<0.05) .The MVD in tumors with positive expression EPCR was increased incomparison with those negative ones. (2) Three recombinant plasmids pshRNA-EPCRwere constructed and sequencing results demonstrated correctly. Compared with H1299and H1299 transfected control plasmid, EPCR mRNA expression surveyed byfluorescent real time RT-PCR decreased markedly in H1299 transfected recombinantplasmid (P<0.01), and similar alterations were found in EPCR protein expression forthese three groups. These results suggest that EPCR expression in H1299 cells could beinhibited efficiently by RNAi. (3) Clonally selected cell of H1299/shRNA strains werescreened in 500μg/ml G418 media. The stable transfection results showed that bothEPCR mRNA and protein were markedly decreased (P<0.05). FCM analysisdemonstrated that H1299/shRNA strains silencing EPCR gene increased accumulationof cells in G0/G1 phase with a decrease in the percentage of cells in S-phase. Cellproliferation, migration and correlated genes such as cyclinE, PCNA and MMP-2 alsoreduced significantly. (4) In invivo experiments of tumor growth, neoplasia formation ofH1299 and xenograft growth were inhibited and life span of host elevated markedly compared with other groups. The MVD in tumors with H1299/shRNA strains was decreased dramatically.Conclusions: (1) EPCR was expressed on many malignant tumor cells and tumor tissues. Although the role of EPCR in cancer biology is poorly understood, it appears that EPCR may contribute to tumor progression. (2)Recombinant plasmid based-shRNA targeting EPCR has an ability to efficiently inhibit EPCR gene replication, expression, block cell proliferation and migration, affect cell cycle and decrease cyclin E, PCNA and MMP-2 in vitro, and diminish tumor growth in vivo. (3) These events in vitro and in vivo suggest a cross-talk between coagulation and carcinoma, and might provide a promising strategy for the treatment of human lung cancer.