Roles And Mechanism Of Netrin4and Neogenin In Fibrosarcoma Cell HT1080

Netrin-4(NTN4) is a new member of the Netrin family in vertebrates,with function of guiding neuron-axonal growth. It not only plays a role in the nervous system, but also has biological functions in many tissues. To view the existing research, NTN4also plays an important role in the development of tumorigenesis. But the role of NTN4on tumorigenesis is still controversial. Some studies have shown that NTN4inhibits tumorigenesis, but some research has proved that it promotes tumorigenesis. And, to view the current research, the reports about NTN4function on tumor invasion and metastasis has been reported rarely, mostly focused on the study of tumor growth and angiogenesis. On the other hand, Neogenin (NEO) is a receptor of axonal guidance factor family and has been reported as a receptor of NTN4. It is low expression in various tumor tissues. At present, whether the function of NTN4is mediated by NEO is still controversial. And so far, no studies on NTN4and NEO interaction on tumor cells.Therefore, their interaction needs to be further studied. The purpose of this paper is to study the effects of NTN4and NEO on tumor cell proliferation, migration and invasion, and further study and discuss whether NEO mediated NTN4function in vitro.HT1080is a highly metastatic human fibrosarcoma cell line, providing an ideal model for studying tumor invasion and metastasis. We found that both NTN4and NEO gene are relatively highly expressed in HT1080cell. Firstly, through the RNAi technology we established NTN4and NEO knock down cell lines. Using these cell lines, we examined the roles of NTN4and NEO in HT1080. Secondly, in vivo experiments using zebrafish tumor explant model to study the metastasis ability of HT1080-NTN4and HT1080-NEO knock down cell lines. In vitro study, we found that knock down NTN4in HT1080promoted cell proliferation, inhibited cell migration and invasion; MMP-9expression decreased; no significant change on MMP-2expression. On the contrary, knocking down NEO in HT1080inhibited cell proliferation, promoted cell migration and invasion; the expression of MMP-2and MMP-9is increased. In addition, in vivo metastasis results are consistent with the in vitro results. However, further study on whether NEO mediated NTN4function in vitro. We found that adding exogenous NTN4protein to the cells can inhibit cell proliferation, promotes cell migration and invasion, But adding same concentration NTN4protein to the NEO knock down cell lines, this function of inhibition and promotion is more significant. And also compared with the NEO knock down cell lines, This function is more obvious. Results show that, NTN4can indeed inhibit the proliferation and promote the migration and invasion of HT1080cells in vitro, and this effect may be mediated by NEO. In addtion, We detected some proteins that related to tumor growth, migration and invasion. We found that knocking down NTN4in HT1080, Vimentin and P-ERK1/2expression decreased, P-p38expression increased; while knocking down NEO in HT1080,Vimentin and P-ERK1/2expression increased, P-p38expression had no significant change.Taken together, we demonstrated that in HT1080cell, NTN4can promote cell migration and invasion, but inhibit cell proliferation. While NEO can inhibit cell migration and invasion, but promote cell proliferation. At least in HT1080cells, NEO and NTN4had opposite roles and different intracellular signaling pathways. In addition, although NTN4and NEO have ligand and receptor relationship in HT1080cells. But this relationship may not be important in many cases. Similarly, NTN4and NEO may also have their own receptor and ligand respectively. In which case, this phenomenon appears and what are the detail molecular events concerning to NTN4and NEO in cells like HT1080is worthy to be further studied.