The Mechanisms Of EGFL7in Inducing EMT To Promote Invasion And Metastasis Of Gastric Cancer Cells

Background:Gastric cancer (GC) remains a major public health burden as one of the most common malignant tumor and the leading cause of cancer-related mortality. Significant progress in the systemic treatment of GC has greatly increased the overall patient survival; due to relapse and metastasis, the five-year survival rate of GC patients remains low. In addition, most newly diagnosed GC patients show metastatic diseases, which still constitute major therapeutic challenges for oncologists.Epidermal growth factor-like domain-containing protein7(EGFL7) is a kind of tumor metastasis related factors, which can significantly enhance cell motility and migration, and is an important sport stimulating factor. EGFL7is an endothelial cell-derived secreted factor that regulates vascular tube formation. Recent studies have reported the expression of EGFL7in several tumors and cancer cell lines, including kidney tumors, malignant gliomas, hepatocellular carcinomas, and colon cancers. We previously demonstrated that EGFL7is also expressed in gastric carcinoma, and its high expression was significantly correlated with pathologic characteristics, clinical progression, poor prognosis, and metastasis. Therefore, EGFL7was suggested as a predictive factor for cancer progression and metastasis. However, the mechanisms underlying its function remains unclear, especially in gastric cancer.Metastasis is a multistep process that triggers the Epithelial-mesenchymal Transition (EMT), in which polarized epithelial cells are converted to mesenchymal cells. EMT initiates the metastatic cascade, which increase cell invasion and migration by conferring the mesenchymal phenotype. Several studies have shown that EMT activation promotes EGF-induced cancer cell migration and invasion. EGFL7, a secreted protein with similarities to EGF, contains two EGF-like domains in its structure. However, through which effector domain of EGFL7affects EMT and promotes cancer metastasis has yet to be determined. Moreover, the molecular mechanisms by which EMT is regulated in GC remain largely unknown.Objective:This study focused on the change of biological behavior of gastric cancer cells and the mechanisms after EGFL7gene silencing and over-expression or deletion domains. The functions of EGFL7were analyzed through the EGFR/protein kinase B (AKT) pathway in GC cells during EMT in order to determine whether EGFL7promotes cancer cell migration/invasion.Methods:1. The study of EGFL7gene silencing on the biological behavior of gastric carcinoma BGC823cell.(1) The expression of EGFL7was investigated by Western-blotting in gastric cancer cell lines:BGC823、SGC7901、MKN28、MKN45and normal gastric cancer mucosa cell line GES-1to identify the cell lines of EGFL7over expression.(2) Constructing interference vector (pGPU6/GFP/Neo-EGFL7-shRNA)and negative control plasmid vector (pGPU6/GFP/Neo-nonspecific-shRNA), and establishing the stable transfected cell line of EGFL7gene silencing.(3) Invasion and migration of gastric cancel cells were detected by cell invasion and migration assay, and scratch wound healing assays.(4) Cell proliferation and growth were investigated by MTT assay and cell colony forming assay.(5) The anoikis of gastric cancer cells were detected by flow cytometry.(6) Xenograft nude mouse model:Tumor growth was evaluated by measuring tumor diameters with a Vernier caliper every3d. The mice were sacrificed at4weeks after cell implantation, and the tumors were extracted and weighed. All livers were examined for metastasis, confirmed by H&E staining. 2. The studies of over-expression and deletion domains of EGFL7on the biological behavior of MKN28gastric cancer cell.(1) The expression of EGFL7was investigated by Western-blotting in gastric cancer cell lines:BGC823、SGC7901、MKN28、MKN45and normal gastric cancer mucosa cell line GES-1to identify the cell lines of EGFL7low expression.(2) Constructing over-expression vector (pGCMV/MCS/IRES/EGFP/Neo-EGFL7+Flag)and deletion domains plasmid vector (pGCMV/MCS/IRES/EGFP/Neo-⊿EMI+Flag, pGCMV/MCS/IRES/EGFP/Neo-⊿EGF+Flag and pGCMV/MCS/IRES/EGFP/Neo-⊿EGF-Ca+Flag), and establishing the stable transfected cell line of EGFL7gene over-expression.(3) Invasion and migration of gastric cancel cells were detected by cell invasion and migration assay, and scratch wound healing assays.(4) Cell proliferation and growth were investigated by MTT assay and cell colony forming assay.(5) The anoikis of gastric cancer cells were detected by flow cytometry.(6) Xenograft nude mouse model:Tumor growth was evaluated by measuring tumor diameters with a Vernier caliper every3d. The mice were sacrificed at4weeks after cell implantation, and the tumors were extracted and weighed. All livers were examined for metastasis, confirmed by H&E staining.3. To explore the mechanisms of EGFL7inducing EMT to promote gastric cancer invasion and metastasis(1) Gastric cancer cell morphology changes were observed after EGFL7gene silencing and over-expression.(2)79gastric adenocarcinoma tissue samples and their corresponding non-neoplastic mucosa specimens were collected, and we detected the expression of EGFL7and EMT-related molecules (E-Cadherin, Vimentin and Snail) by immunohistochemical method and also analyzed the correlation among them.(3) The mRNA level of the EMT-related molecules (E-Cadherin, Vimentin and Snail), invasion and metastasis(MMP-9), angiogenesis(VEGF) related indicators were detected by Real-Time PCR.(4) The protein level of the EMT-related molecules (E-Cadherin, Vimentin and Snail), invasion and metastasis(MMP-9), angiogenesis(VEGF) related indicators were verified by Western-blotting.(5) The protein level of the EMT-related molecules (E-Cadherin, Vimentin and Snail), invasion and metastasis(MMP-9), angiogenesis(VEGF) related indicators and the total expression and phosphorylation of EGFR, ERK and AKT were all assayed by Western-blotting after EGF receptor (EGFR) inhibitor AG1478treatment.Results:1. EGFL7gene silencing could reduce the invasion and metastasis of gastric cancer cells(1) BGC823had the highest expression of EGFL7in gastric cancer cells; EGFL7gene silencing stable gastric cancer cell lines was established successfully.(2) EGFL7gene silencing could induce anoikis of gastric cancer cells, which could inhibit the invasion and metastasis of gastric cancer cells, but not affect gastric cancer cell proliferation and growth.(3) Compared with the control group, EGFL7gene silencing group had the reduction in the number of liver metastases in nude mice.2. The over-expression of EGFL7could enhance invasion and metastasis of gastric cancer cells, EGF domain is the effector domain of EGFL7(1) MKN28had the lowest expression of EGFL7in gastric cancer cells; EGFL7over-expression and deletion domains stable gastric cancer cell lines were established successfully.(2) EGFL7over-expression could increase gastric cancer cell resistance to anoikis, which could promote invasion and metastasis of gastric cancer cells, but not affect gastric cancer cell proliferation and growth.(3) After EGF domain deletion of EGFL7could increase anoikis of gastric cancer, which could inhibit invasion and metastasis of gastric cancer cells, indicating that EGF domain was the effector domain of EGFL7. (4) Compared with the control group, the over-expression of EGFL7group in nude mice had an increase in the number of liver metastasis.3. EGFL7promote EMT of gastric cancer cells through phosphorylation of AKT(1) The cell morphology of expressing high EGFL7(BGC823, BGC-NC and MKN28-EGFL7) displayed loss of intercellular contact and a spindle-shape, typical of the mesenchymal phenotype, whereas that of expressing low EGFL7(BGC2-13, MKN28, and MKN28-NC) cells exhibited small cell size, cobblestone-like shape, and tightly arranged intercellular contact, typical of epithelial cell-like forms.(2) EGFL7expression was positively correlated with Vimentin (r=0.620, P<0.05) and Snail (r=0.492, P<0.05) levels, and negatively correlated with E-Cadherin expression (r=-0.304, P<0.05)(Spearman’s rank correlation coefficient)(3) EGFL7gene silencing could reduce the level of EGFR and AKT phosphorylation.(4) EGFL7gene silencing could inhibit the expression of Vimentin, Snail, MMP-9and VEGF, and promote the expression of E-Cadherin.(5) EGFR inhibitor could reduce the level of EGFR and AKT phosphorylation.(6) EGFR inhibitor could inhibit the expression of Vimentin, Snail, MMP-9and VEGF, and promote the expression of E-Cadherin.Conclusion:1. EGFL7gene silencing could reduce gastric cancer cells anoikis resistance, which reduced the invasion and metastasis of gastric cancer cells, but not affect gastric cancer cell proliferation and growth.2. EGF domain is functional role played in EGFL7gene.3. EGFL7was an important regulator of a characteristic metastatic process in GC cells, and its over-expression played a key role in gastric tumorigenesis and metastasis. Moreover, we had demonstrated for the first time the EGFL7promotes metastasis by activating EMT through an EGFR-AKT-Snail signaling pathway. Disruption of EGFL7-EGFR-AKT-Snail signaling may a promising therapeutic strategy for gastric cancer. Figures37；Tables7；Refernces142.