| Backgroud Lung cancer is the greatest threat to human health and life with fastest growing morbidity and mortality in the contemporary world. Metastasis is the sign and character of malignance in lung cancer, and also the major reason of treatment failure and patients’death, about 30% of patients are diagnosed with distant metastasis at first visit, besides,50%-60% of patients develope metastatic tumors in the therapeutic process.80%-90% of lung cancer-related death are caused by metastases[1,2]. In the recent years, application of Chronobiology in the field of lung cancer research make a new road for revealing molecular mechanisms of lung cancer metastases. Circadian clock genes are not only involved in the regulation of normal cells’biological behavior, but also influence cancer cells’behavior, suggesting that they may be a tumor suppressor gene. Of late years, biological clock genes especially period genes have become a new focus of domestic and overseas lung cancer research field[3-5]. There were researches suggesting that Per2 participate cancer occurrence and development and cell apoptosis. CDH1,Nm23,KAI1 and MTS1 are tumor suppressor genes currently known, while c-myc, VEGF and CD44 are tumor-promoting genes. However, whether Period genes regulate lung cancer metastasis or not have not been studied yet.Objective To clarify the effect and significance of Per2 gene in the invasion and metastasis of lung cancer.Methods In vitro assays:Using lentivirus transfected A549 lung cancer cells with eukaryotic expression vector pcDNA3.1 (+)-Per2 to construct stable cells, varified Per2 gene overexpression with RT-PCR and Western Blotting. Improved Boyden cell method and Wound Healing assay were used to detect in vitro invasion and metastasis ability of above-mentioned cells. RT-PCR and Western Blotting were performed to detect the expression level of NM23, VEGF, c-myc, CDH1 and CD44 genes and proteins. In vivo assays:Per2, Nm23 and CDH1 expression in the subcutaneous transplantation tumor were observed with immunohistochemical technique. Inoculate the nude mouse with stable transfected A549 cells through tail vein, construct invasion and metastasis animal model of A549 lung cancer cells.Anatomy brain, lung, liver and adrenal gland organization in the two groups of nude mice with HE staining and counting.Results In vitro assays:RT-PCR validated that Per2 of OE(over-expressed) group were 11 times higher than NC(negative control) group. Western blotting showed the same results, and Gray scale quantitative confirmed the statistical difference(p=0.0425). Transwell invasion and migration assay manifested that the number of NC cells pass through the chamber were 134.2±7.165 and 130.8±9.987, while the number of OE group were 56.80±6.763 and 20.20±4.164, experimental group were significantly weaker than control group (p< 0.0001 and p<0.0001, respectively). Wound healing assay showed that the mean migration distance of NC group were 452.8±5.279,while OE group were 315.4±5.743, the difference was statistically significant(p<0.0001). RT-qPCR and western blotting demonstrated that expression level of VEGF, CD44 and c-myc were lower in transfected cells(OE) than NC cells,while CDH1 and Nm23 were higher than the latter with statistical significance(p<0.05). In vivo assays:Immunohistochemical staining of subcutaneous tumor indicated that Nm23 and CDH1 expression in OE group were higher than NC group (p=0.0382 and p=0.0232 respectively).Conclusion Per2 gene inhibit tumor cells invasion and migration dramatically. Per2 influence tumor progress through regulation of metastases associated genes. Among which tumor-promoting genes VEGF,CD44 and c-myc were down-regulated, while tumor suppressor genes such as Nm23 and CDH1 were upregulated. Our work lay the foundation for mechanism research of Per2 influence lung cancer invasion and metastasis, possessing great theoretic significance in developing a new way to diagnosis and treatment of lung cancer metastases. |
