TIPE1 Inhibits The Invasion And Migration Of Gastric Cancer Through Regulating EMT

OBJECTIVE:Gastric carcinoma (GC) has high morbidity and mortality in the worldwide as a common digestive tract malignant tumor. According to the latest data of the world health organization (WHO) in the world cancer report, the incidence of gastric cancer of our country has become the top of the world accounting for 42% of the global total cases. Finding the key markers for early clinical diagnosis of gastric cancer and further finding potential targets for gastric cancer treatment are the hot research fields in recent years.A common characteristic of GC is early invasiveness into surrounding tissues and the peritoneal cavity. The malignant progression of GC is the main reason for poor prognosis. Therefore, it is urgent to find the key molecular of the progression in GC. TIPE1 (TNFAIP8-like1) is a new member of the TIPE8 (tumor necrosis factor-a-induced protein 8 or TNFAIP8) family which are closely related to cell apoptosis, signal transduction, proliferation, invasion and metastasis of tumor cells. The TIPE family (TNFAIP8, TIPE1, TIPE2 and TIPE3) plays an important role not only in immune homeostasis but also in cancer. Previous studies have shown that TIPE1 has an important role in cell death and could induce apoptosis. However, most of its functions is unclear and need to be investigated. In this study. we focus on the potential role of TIPE1 in GC progression and metastasis.METHODS:1. The expression of TIPE1 in clinical specimens of GC1.1. The 100 patients were diagnosed with primary gastric cancer. Real-time RT-PCR and immunohistochemistry (IHC) were used to detect the expression of TIPE1.1.2 100 cases of clinical pathologic data were obtained in hospital pathology department, analysis the correlation between TIPE1 expression and clinical pathological stage.2. The role and mechanism of TIPE1 in the progression of GC2.1 We over express TIPE1 using lentivirus in SGC7901 cells and BGC823 cells and knock down TIPE1 expression using TIPE1 specific RNAi in AGS.2.2 We observe the change of GC cell migration and invasion after over expressing TIPE1 through wound healing and transwell in vitro.2.3 We then examine whether TIPE1 overexpression inhibited the metastasis of gastric cancer cells in vivo. We injected BGC-823 cells and SGC7901 cells transfected with lentivirus-con or lentivirus-TIPE1 into the tail vein of nude mice and examined their lungs for tumor seeding.2.4 We observed the change of EMT related maker E-cadherin, Vimentin, Slug, Snail, Twist and MMP2, MMP9, β-catenin after overexpression or silence TIPE1 through Real-time RT-PCR, western blot and confocol.RESULTS:1. The expression of TIPE1 was inversely correlated with differentiation status and distant metastasis in primary gastric cancer tissuesTo evaluate the prognostic performance of TIPE1 expression in subgroups of GC. a stratification analysis was conducted. We collected 100 cases of primary gastric cancer specimen. All of the eligible GCs were stratified by TNM stage and histological grade. TIPE1 expression was inversely correlated with differentiation status (P=0.000), lymph node status (P=0.039), distant metastasis (P=0.003) and TNM stage (P=0.025) of patients with gastric cancer. We also measured TIPE1 expression in paired normal and tumor samples from 100 GC patients using Real-time RT-PCR and IHC. As expected, we found that high-expression level of TIPE1 was associated with high degree of GC cell differentiation and effectively predicted free of distant metastasis.2. TIPE1 inhibited migratory and invasive capacities of GC cell lines in vitroBoth SGC7901-TIPE1 and BGC823-TIPE1 cells had significantly slower closure of the wound area compared to their control cells. This result was confirmed by Boyden’s chamber assay. Moreover,SGC7901-TIPE1 and BGC823-TIPE1 cells showed a lesser degree of invasion through Matrigel.3. TIPE1 inhibited the migration and invasion of gastric cancer cells in vivoWe injected BGC-823 cells and SGC7901 cells transfected with lentivirus-con or lentivirus-TIPE1 into the tail vein of nude mice and examined their lungs for tumor seeding. Metastatic nodules were fewer with lentivirus-TIPE1 than lentivirus-con group.4. TIPE1 suppressed the expression of molecular markers associated with EMTThe high-expression level of TIPE1 in well-differentiated gastric tumors suggested that TIPE1 expression may be associated with degree of tumor cell differentiation. To test this, we surveyed the expression of TIPE1 in a panel of GC cell lines that are derived from tumors with various degrees of differentiation. TIPE1 expression was down-regulated in poorly differentiated GC cell lines compared with well-differentiated GC cell line. EMT is a vital biological process involved in cell differentiation in normal embryonic development and is a potential mechanism for tumor cell metastasis. We find that over-expression of TIPE1 significantly altered the expression of molecular markers associated with EMT.The epithelial cell markers such as E-cadherin (E-cad) were up-regulated compared with the control groups, whereas mesenchymal cell marker vimentin and transcription factors that are known to promote EMT such as snail, slug, and twist were down-regulated. Furthermore, we knocked down the expression of TIPE1 in AGS cells using TIPE1-specific RNAi. Approximately 70% of TIPE1 were down-regulated in TIPE1 RNAi-transfected cells compared with control RNAi-transfected and RNAi-nontransfected AGS cells. The epithelial cell markers E-cad were down-regulated in TIPE1 RNAi-transfected cells, whereas mesenchymal cell markers vimentin and EMT-promoting transcription factors were up-regulated. Up-regulation of E-cadherin and down-regulation of vimentin in TIPE1-overexpressed BGC823 cells were further confirmed by confocal. Since overexpression of TIPE1 in GC cells leads to transcriptional changes of several markers involved in EMT, we hypothesized that it may interact and/or regulate a common factor that is upstream of those EMT markers. To test this hypothesis, we investigated whether TIPE1 regulates the expression of β-catenin. β-Catenin is a crucial regulator in the Wnt signaling pathway. Most of the EMT-associated molecules such as slug, twist, and MMPs are directly or indirectly regulated by P-catenin. We measured the β-catenin, MMP2 and MMP9 expression level in SGC7901 and BGC823 cells treated with/without TIPE1-expression lentivirus. Significant differences of β-catenin, MMP2 and MMP9 protein expression were observed among different experimental groups.Collectively,TIPE1 plays an important role in regulating EMT marker expression in gastric cancer cells.CONCLUSION:1. The expression of TIPE1 in GC tissue decrease significantly. And it is closely related to the differentiation degree and the TNM status of GC.2. TIPE1 can significantly inhibit the migration and invasion of GC cells in vitro and in vivo.3. TIPE1 inhibits the migration and invasion of GC cells mainly through EMT signaling pathway.