The Expression Of Interferon-induced Transmembrane Protein 3 In Gastric Cncer And Its Related Mechanism

[Background] Gastric Cancer is one of the highest cancer incidence and mortality in the world. According to the statistics, there are over 750,000 new cases diagnosed annually worldwide, and the overall 5-year survival rate remains below 25%. Currently, early detection, early diagnosis and early treatment are quite important measures to enhance the survival rate of gastric cancer in the early stage. Therefore, finding the new therapeutic targets and early diagnosis biomarkers of gastnc cancer is key to controlling the initiation and progression of GC.Interferon-induced transmembrane protein 3 (IFITM3), also known as 1-8U, is a member of the IFN-inducible transmembrane protein family. It consists of short, two transmembrane domain proteins (5-18 kDa) with a high core sequence similarity but divergent N-and C-termini. Moreover, the IFITMs play an important role in different cell processes, including cell adhesion, immune-cell regulation, germ-cell homing and maturation, and bone mineralization.[Objective] This study is aimed to determine the expression levels of IFITM3 in 48 cases of gastric cancer tissues and cells, and by carrying outin vitro experiments to detect the effects of IFITM3 to various aspects of the malignant phenotype of human GC,including cell migration, invasion, proliferation and cell cycle, which reveals the function and molecular mechanisms of IFITM3 in gastric cancer preliminary.[Methods] 1. The mRNA expression levels of IFITM3 were tested by quantitative real-time PCR (qRT-PCR) for detecting the collected specimens of 48 cases of gastric cancer and further relationship between the expression of IFITM3 and the clinicopathological factors was analysed; In addition, the expression of IFITM3 levels in GC cell line AGS, SGC7901, BGC823, MKN45, MKN28 and human gastric epithelial mucosa cell line GES-1 were detected by qRT-PCR and western blot; The IFITM3 protein levels of gastric cancer tissues in different pathological stages were detected by immunohistochemistry.2. Stably transfected gastric cancer cell lines of IFITM3—downregulated was established by lentivirus construction successfully, and the effect of IFITM3 on cell migration, invasion and proliferation in vitro were detected by wound healing assays, transwell membrane assays, CCK-8 assays and flow cytometry.3. Changes in cell morphology were observed under inverted microscope before and after the IFITM3 expression was changed; The expression at the RNA and protein levels of EMT associated gene(E-Cadherin and Vimentin) in IFITM3-silence, negative control and normal GC cells were detected by qRT-PCR and Western blot, respectively.4.The different levels of MMP-2 and MMP-9 expression between the IFITM3-silence and normal gastric cancer cells were detected by qRT-PCR and Western blot.5. After using Wnt/β-catenin pathway inhibitor XAV939 in gastric cancer cell lines with different concentrations, qRT-PCR and western blot were detected the change of the expression of β-catenin and IFITM3.[Results] 1.IFITM3 levels were markedly upregulated in cancer tissues compared with corresponding non-cancer tissues. The potential related factors affecting IFITM3 expression were determined and it was confirmed that IFITM3 expression was significantly associated with tumor differentiation, lymph node and distant metastasis, and TNM stages by the clinical and pathological data analysis. Compared with human gastric epithelial mucosa cell line GES-1, IFITM3 expression in SGC-7901, BGC-823 and AGS cells were significantly higher, while no significant difference were found in MKN28 or MKN45 cells.2. Stably transfected cell lines of IFITM3-silence were established successfully, CCK-8 assays showed that IFITM3 overexpression contributes to the proliferation of gastric cancer cells, Depletion of IFITM3 arrested tumor cells at the G0/G1 phase and reduced the cell numbers in the S phase of the cell cycle.3. Wound healing assays and transwell assays confirmed that IFITM3-silence significantly suppressed the migration and invasion of GC cells. 4. Under an inverted microscope, IFITM3 knockdown cells induced round spheroids with no or few protrusions. The increased expression of E-cadherin and decreased expression of vimentin was detected in IFITM3 knockdown cells in the mRNA and protein levels.5. MMP-2 and MMP-9 were significantly downregulated in IFITM3-silence GC cells. MMPs are involved in IFITM3-induced cell migration and invasion.6. The expression of P-catenin and IFITM3 maintained a high level in normal GC cells. After incubating with XAV939 with 5 and 10μ/mol, IFITM3 expression markedly decreased together with the reduction of P-catenin by Western blot and qRT-PCR.[Conclusion] In gastric cancer tissues, the high expression of IFITM3 is closely related to tumor differentiation, lymph node and distant metastasis. IFITM3 can be used as a tumor-inducing factor directly affects the biological characteristics of cell migration, invasion and proliferation. IFITM3 can be a potential new molecular marker and target for the gastric cancer diagnosis and treatment.