Proteins from cell and tissue samples were separated by SDS\PAGE and electrotransferred onto PVDF membranes (Millipore). and rescue assays. Consequently, our results indicated that ANTXR1 induced proliferation, cell cycle progression, invasion Necrostatin 2 and migration, and tumorigenicity and induced suppressed apoptosis in GC. Mechanistic investigation indicated that ANTXR1 exerted its promoting Rabbit polyclonal to Zyxin effects on GC through activation of the PI3K/AKT/mTOR signaling pathway. In conclusion, our findings suggested that ANTXR1 plays a crucial role in the development and progression of GC and could serve as a novel prognostic biomarker and potential therapeutic target for GC. gene.8 Tumor endothelial marker 8 is a highly conserved cell\surface glycoprotein that was originally identified by its overexpression in ECs that line the tumor vasculature of colorectal cancer.8 Several studies have shown that TEM8 binds to the C5 domain of collagen type VI and promotes migration of ECs in vitro.9, 10 Furthermore, TEM8 plays a significant role in cell attachment and migration, and interacts with ECM proteins and the actin cytoskeleton. It also mediates adhesion of cells to type 1 collagen and gelatin, reorganization of the actin cytoskeleton, and promotes cell spreading.11 Previous studies found that TEM8 is involved in the angiogenic response of cultured umbilical vein ECs by regulating cellCmatrix interactions on collagen.12 Originally, TEM8 was identified as one a cell surface receptor of anthrax toxin, so it was alternatively named ANTXR1.13 Recent studies identified ANTXR1 as the high\affinity cellular receptor for SVV.14 Seneca Valley virus has shown encouraging results and a favorable safety profile as an oncolytic virus in clinical trials, and this finding offers a promising biomarker for selecting patient response to treatment.11, 15, 16, 17 The extracellular domains of ANTXR1 share homology with integrins, and interactions with collagen IV, collagen VI, and laminin suggest a possible function in basement membrane assembly and angiogenesis.18, 19 In comparison with the wide distribution in normal tissue of ANTXR2, ANTXR1 is overexpressed in tumor cells and the vasculature of developing carcinoma.9, 12 Previous studies reported that approximately 63% of cell lines surpass the expression cut\off line of ANTXR1 among 1037 cell lines in the Cancer Cell Line Encyclopedia.14 In the present study, we found that ANTXR1 plays a critical role in promoting GC progression. A series of in vitro and in vivo assays revealed that knockdown of ANTXR1 in GC cells dramatically Necrostatin 2 suppressed cell proliferation, Necrostatin 2 cell cycle progression, invasion and migration, and tumorigenicity and induced apoptosis, whereas overexpression of ANTXR1 had the opposite effect. Furthermore, our mechanistic investigations revealed that ANTXR1 induced GC cell proliferation and aggressiveness by activating the PI3K/AKT/mTOR signaling pathway. Our findings indicated that plays a role as a novel oncogene in GC and could be a potential diagnostic and therapeutic target. 2.?MATERIALS AND METHODS 2.1. Tissue specimens Human GC tissue and adjacent nonmalignant tissue were obtained from the Department of General Surgery in Xinhua Hospital affiliated with Shanghai Jiao Tong University (Shanghai, China). None of the patients received radiotherapy or chemotherapy before surgery. All diagnostic information was gathered based on the American Joint Committee on Cancer (8th edition) guidelines. We obtained informed consent from all patients and the study was approved by the Research Ethics Committee of Xinhua Hospital, School of Medicine, Shanghai Jiao Tong University (approval no. XHEC\F\2019\029). 2.2. Cell lines and reagents The 4 human GC cell lines, BGC823, MGC803, HGC27, and SGC7901, and human gastric mucosal epithelial cell line (GES\1) were purchased from the cell bank of the Chinese Academy of Sciences (Shanghai, China). All cells were authenticated by short tandem repeat profiling and cultured in RPMI\1640 (Hyclone) containing 10% FBS (Gibco). These cell lines were incubated in a humidified incubator containing 5% CO2 at 37C. LY294002 (Cat. No. 154447\36\6) was purchased from MedChemExpress and dissolved in DMSO. Both HGC27/ANTXR1 and SGC7901/ANTXR1 cells were treated with LY294002 for 48?hours at the recommended concentration of 20?M. 2.3. Total RNA extraction and qRT\PCR Total RNA was extracted from the cells or tissue samples using TRIzol reagent (Invitrogen). The cDNA was synthesized using the PrimeScript RT reagent kit with gDNA Eraser (TaKaRa) according to the manufacturer’s instructions. Quantitative real\time PCR was undertaken using SYBR Green (TaKaRa) according to the manufacturer’s instructions. Target genes were measured by using an Applied Biosystems StepOnePlus real\time thermocycler (Applied Biosystems). The specific primer sequences used to amplify ANTXR1 were: 5\ACAGTTGGCTCACAAATTCATCA\3 (forward) and 5\ TCACTGGCCCTTTCAAATCCT\3 (reverse). GAPDH was used as an endogenous control in PCR analysis. Relative gene expression levels were quantified by the comparative 2?Ct method and cycle thresholds were normalized to GAPDH levels. 2.4. Western blot analysis The protein was extracted by RIPA lysis.