| ObjectiveGlobally, lung cancer is the leading cause of death in male and is the second leading cause of death in famale. In recent years, the mortality of lung cancer is growing very quickly. Non-small-cell lung cancer(NSCLC), including lung squamous cell carcinomas, lung adenocarcinoma and large cell lung cancer, accounts for about 85% of total number of lung cancer. Although diagnosis and treatment of the diseases are developing rapidly, five-year survival rate of non-small-cell lung cancer is still low. The discovery and research of oncogenes and suppressor genes are very helpful to the diagnosis and treatment of cancer. Therefore, To search for the genes that play important roles in non-small cell lung cancer makes great sense and to study its mechanism is of far-reaching significance.TIPE3 is the newest member of the TNFAIP8 family. Professor Youhai Chen in University of Pennsylvania collaborated with our laboratory, first discovered that TIPE3 is a cancer promoting molecule. TIPE3 can act as a phosphoinositide carrier that promotes the proliferation of tumor cells. The organs that express TIPE3 in human and mice are nearly identical, though there are some differences. Human TIPE3 and mice TIPE3 are prior expressed in the epithelial originated cells with secretion function. In many tumor cell lines, TIPE3 is highly expressed. The N-terminal domain of mice TIPE3 maintains the function of cell proliferation and survival. The crystal structure of TIPE3 shows that it is composed of seven alpha helixes. These alpha helixes construct a hydrophobic cavity which is associated with the combination of phosphoinositides. TIPE3 can bind lipids through its TH domain. Moreover, TIPE3 can bind many kinds of phosphoinositides, including PIP2 and PIP3. In terms of molecular mechanism, TIPE3 mainly activates PI3K-AKT and MEK-ERK signaling pathways. In our previous study, we discovered that TIPE3 which was highly expressed in non-small cell lung cancer patients can form two kinds of TIPE3--cell membrane localized TIPE3 and cytoplasm localized TIPE3. Cell membrane localized TIPE3 is associated with poor TNM stage compared with cytoplasmic TIPE3. Thus, we hypothesized that the localization of TIPE3 in cells may be related to its function, playing a key role in the evolution of non-small cell lung cancer. At present, the function and mechanism of human TIPE3 in NSCLCs are yet to be reported, which is the purpose of this research.In order to determine the TIPE3 function in non-small cell lung cancer, this paper intends to study in the following aspects:First, use in vivo and in vitro assays to detect TIPE3’s role in the cell proliferation and migration in non-small cell lung cancer. Second, make it clear whether the localization of TIPE3 relates to its function. Third, explore the molecular mechanism of TIPE3.MethodsⅠ. The effect of TIPE3 in non-small cell lung cancer on cell proliferation and migration In vivo and in vitro.1. Endogenous expression of TIPE3 was detected in A549 and H1975 by RT-PCR and Western blot.2. By knocking down TIPE3 in H1975 cells, we detected cell proliferation by CCK8 assay. Transwell assay is applied to detect cell migration.3. In A549 cells that transfected with TIPE3 lentivirus, we tested its transfection efficiency. CCK8 and Transwell assays were applied to detect cell proliferation and migration. Moreover, to get more evidences that TIPE3 promotes cell migration, we knocked down TIPE3 in A549 cell line which was already transfected with TIPE3 lentivirus.4. We transfected TIPE3 plasmids to H1975 and A549 cell lines in order to detect the cell proliferation through CCK8 assay. We also inspect the cell migration through transwell assay.5. We transfected TIPE3 lentivirus to A549 cells and established the subcutaneous xenograft tumor model, in order to detect the effect of TIPE3 on tumor growth in vivo. We drew the growth curve of the xenograft tumor. The mice were sacrificed and the tumor was weighed at last.Ⅱ. The localization of TIPE3 relates to its function.1. Through immunofluorescence assay, we tested the endogenous TIPE3 expression in H1975 and A549 cell lines.2. We applied the immunofluorescence assay to detect the localization of TIPE3 in A549 after TIPE3 is overexpressed by plasmid or lentivirus, and we compared the localization with the function.3. We prepared the paraffin sections of the subcutaneous xenograft tumor. We observed the localization of TIPE3 through immunohistochemistry method. Then we compared the localization with the function.III. The molecular mechanism of TIPE31. We applied Western blot method to detect PI3K-AKT and MEK-ERK signaling pathways after TIPE3 plasmid or lentivirus was transfected.2. We knocked down TIPE3 after A549 cells are stably transfected with TIPE3 lentivirus, in order to detect PI3K-AKT and MEK-ERK pathways.3. The activity of Rac1 was examined in a549 cells that are stably transfected with TIPE3 lentivirus.Results1. The endogenous TIPE3 is expressed in both cell membrane and cytoplasm, and TIPE3 mainly gathers in the cell membrane in the cells with high viability.To search for the TIPE3 function in NSCLC, A549 and H1975 cell lines were introduced and their endogenous TIPE3 were detected by RT-PCR and Western blot. We found that at both genetic and protein level, TIPE3 was highly expressed in H1975 cell line while lowly expressed in A549. The immunofluorescence certified this result. Notably, we discovered that the endogenous TIPE3 was highly expressed in the cell membrane of A549 and H1975 which had many pseudopodia, showing their high viability.2. The cytoplasm localized exogenous TIPE3 inhibit the cell growth and migration.We constructed the long and short TIPE3 plasmid with N-terminal flag tag. When transfected with two plasmids TIPE3, the cell growth and migration were restricted. To find out the reason, we applied immunofluorescence assay to detect the localization of TIPE3. The results showed that although short TIPE3 was highly expressed, even in the cells with many pseudopodia and high viability, we hardly saw membrane localization of TIPE3. Therefore, we speculated that TIPE3 function may be associated with its localization.3. Cell membrane localized endogenous TIPE3 can promote cell proliferation and migration.We further applied the TIPE3 siRNA to detect cell proliferation and migration by CCK8 assay and transwell assay, respectively. We discovered that knocking down TIPE3 effectively inhibited the cell growth and migration, showing the promoting effect of TIPE3 on NSCLCs.Then we constructed TIPE3 lentivirus and transfected it to A549 cell line. The stably expression of TIPE3 in A549 cells has faster cell growth and migration compared to the control. Then we knocked down TIPE3 in A549 with stably expressed TIPE3. The cell migration ability was inhibited. Besides, the immunofluorescence showed TIPE3 mainly gathered in the form of point in the membrane. These results indicated that cell membrane localized exogenous TIPE3 can promote cell proliferation and migration.4. Cell membrane localized exogenous TIPE3 can promote cell proliferation in vivo.In order to complete our point of view in vivo, we established the subcutaneous xenograft tumor model in nude mice. We discovered that TIPE3 tumors grew more quickly than control. Besides, the immunohistochemistry showed that TIPE3 was mainly localized in the cell membrane of tumors. These results suggested that cell membrane localized TIPE3 can promote cell proliferation in vivo.5. The membrane localized TIPE3 promotes PI3K-AKT and MEK-ERK signaling pathways, while cytoplasm localized TIPE3 inhibits them.We explored the molecular mechanism of TIPE3 that affect cancer. p-ERK and p-AKT levels had been upregulated when TIPE3 is membrane localized by Western blot. But when cells are transfected with short TIPE3, p-ERK and p-AKT levels are downregulated. These results indicated that TIPE3 played its role through PI3K-AKT and MEK-ERK signaling pathways. Besides, we also discovered that membrane localized TIPE3 promotes the Racl activity, it could be another mechanism for TIPE3.Conclusion1. TIPE3 localization is associated with its function. Membrane localized TIPE3 can promote cell growth and migration.2. TIPE3 plays its role through PI3K-AKT and MEK-ERK signaling pathway.Innovation1. We put forward TIPE3 functions related to its subcellular localization for the first time. It is helpful for further research of function of TIPE3.2. For the first time, we discovered that the membrane localized TIPE3 promotes PI3K-AKT and MEK-ERK signaling pathways, while cytoplasm localized TIPE3 inhibit them. The new mechanism is revealed.Limits1. We did not fully explain why the cytoplasm TIPE3 causes inhibitory effect on cell growth and migration.2. We illustrate the relationship between localization and function only by immunofluorescence assay. We need to perform more assays to prove it. |
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