| Background and ObjectiveGastric cancer (GC) is the most common malignant tumor of digestive system and its incidence and mortality rank the forth and the second among the common malignancies worldwide, respectively. The incidence and mortality of GC ranks the first among digestive tract malignant tumors in China. The incidence of GC varies greatly in different regions and this is caused by the differences in the distribution of the main risk factors. The risk factors of GC include Helicobacter pylori infection [6], bad living habits (such as smoking, drinking alcohol, taking too much smoked, salted and pickled foods), chronic atrophic gastritis and gastric ulcer. GC includes a variety of pathological types, among which, gastric adenocarcinoma accounts for 90% of the total number. Gastric adenocarcinoma is the main research objective of our team. Because of the characteristics of occult onset, high degree of malignancy, rapid development and prone to metastasis, early diagnosis rate of GC is less than 10% and the prognosis of advanced GC is poor. Although great progress has been made in diagnosis and treatment of GC, operation is still the most effective method to cure GC at present. Multiple factors, stages and genes are involved in the occurrence, development and metastasis of GC, therefore, studying gene mutations and the change in factors those play roles in this process will help to determine the biological behavior of GC and evaluate the prognosis of patients. It is very important to screen effective biomarkers for early diagnosis, treatment and prognosis evaluation of GC.Tumor necrosis factor-alpha-induced protein (TNFAIP) family are proteins those induced by tumor necrosis factor a (TNF a) and are involved in many biological processes through activating nuclear factor-kb (NF-kb). TNFAIP8 belongs to TNFAIP family and includes TIPE (TNFAIP8), TIPE1 (TNFAIP8L1), TIPE2 (TNFAIP8L2) and TIPE3 (TNFAIP8L3). TNFAIP8 contains a death domain (DED) and plays roles in the regulation of cell proliferation, inflammation, apoptosis, etc. Studies show that the expression of TNFAIP8 in many human solid tumors is correlated with tumors occurrence, development, invasion, metastasis and prognosis. Over expression of TNFAIP8 in tumor cells can enhance cell proliferation and promote the growth of tumor and cancer characteristics of tumor cells are significantly reduced when TNFAIP8 gene is knocked out. Thus, these observations demonstrated that TNFAIP8 may play a key role in tumor invasion and metastasis and and may be a potential marker for clinical evaluation of tumor aggressiveness and prognosis.So far, there is rare study on the relationship between TNFAIP8 expression and clinical pathological features of gastric adenocarcinoma and the relationship between TNFAIP8 expression and prognosis of gastric adenocarcinoma is not clear. TNFAIP8 siRNA was synthesized and transfected into SCG7901 gastric cancer cell line, and the effect of interference of TNFAIP8 expression on migration and invasion of SCG7901 cell was evaluated. To further investigate the possible mechanism of TNFAIP8 involved in invasion and metastasis.Methods1. The expression of TNFAIP8 in Gastric adenocarcinoma:TNFAIP8 expression in 106 gastric adenocarcinoma, paracancerous tissues and corresponding metastatic lymph nodes were detected by immunohistochemical method. The correlation of TNFAIP8 expression with clinical pathological data and gastric adenocarcinoma prognosis was analyzed..2.Synthesis of TNFAIP8 siRNA and transfection:Western Blot were used to detect the expression of TNFAIP8 gene in gastric cancer cell line of SGC7901,MGC803, BGC823, and select the highest expression one. Three kinds of siRNA (TNFAIP8 siRNAl, TNFAIP8 siRNA2 and TNFAIP8 siRNA3) were synthesized to interference the gene of TNFAIP8, Gene transfection were used to transfer the siRNAs into the gastric cancer cell line. Then flow cytometry was used to observe the transfection efficiency. Real-time PCR and Western Blot were used method to detect the expression of protein and mRNA of TNFAIP8 after the interference.3.Effect and role of TNFAIP8 on metastasis of gastric cancer:Select the group that has the highest efficiency of silence the TNFAIP8. Treadmill test was used to detect cell-forming ability of monoclonal. CCK-8 was used to detect the cell proliferation, and the transwell chamber was used to detect cell migration and invasion. In order to understand the possible mechanism of TNFAIP8 involved in the process of metastasis, western blot were used to detect the difference of expression of gastric cancer related gene(P53, MMP-2, MMP-9) in the level of RNA and protein before and after interference.Results1.The high expression of TNFAIP8 in adjacent normal gastric mucosa, gastric adenocarcinoma and metastatic lymph node successively increased, with the rate of 0,47.2%(50/106) and 81.7%(49/60), respectively. There was statistically significant difference between each two groups (P< 0.05). TNFAIP8 high expression rate in the primary tumors (60%,36/60) with lymph node metastasis was significantly higher than those without lymph node metastasis (30.4,14/46) (P < 0.05).2.TNFAIP8 expression was related to TNM stage, T grade, vascular invasion, lymph node metastasis and the level of serum CA72-4. Survival analysis showed that the overall survival rate of gastric adenocarcinoma patients with high expression of TNFAIP8 was poorer than patents with low TNFAIP8 expression and TNFAIP8 expression was negatively associated with the prognosis.3. Multivariate Cox model analysis showed that the expression level of TNFAIP8 was an independent prognostic marker in gastric adenocarcinoma (RR 1.736, P=0.029).4.SiRNA transfection:western blot results showed that TNFAIP8 gene was most highly expressed in SCG7901 than in MGC803 and BGC823. Group TNFAIP8 siRNA2 had the highest silencing efficiency on interfering the expression of TNFAIP8. The expression of TNFAIP8 in RNA and protein levels were significantly decreased after transferred with siRNA.5.Effect and role of TNFAIP8 on metastasis of gastric cancer:After the interference of TNFAIP8 gene, the ability of Monoclonal cell formation, cell proliferation, migration ability and invasion of SCG7901 were significantly decreased, and the expression of MMP-2, MMP-9 were reduced in protein levels.Conclusion1.High TNFAIP8 expression was detected in gastric adenocarcinoma. Moreover, the high expression rate of TNFAIP8 in LNM was higher than that in primary tumors.2.The expression level of TNFAIP8 was positively correlated with the invasion and metastasis of gastric adenocarcinoma, and negatively correlated with the prognosis. TNFAIP8 could be a prognostic marker and a potential therapeutic target of gastric adenocarcinoma.3.TNFAIP8 siRNA could inhibit the expression of TNFAIP8 in SCG7901 cells. TNFAIP8 gene could promote the proliferation, migration and invasion of gastric cancer cells, and reduce the expression of gastric cancer-related gene. It plays an important role in the development and pathogenesis of the gastric cancer. |
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