The Role And Mechanism Of TRIB3 In Promoting The Invasion And Metastasis Of Gastric Cancer

Background:Gastric cancer(GC)is one of the most common gastrointestinal malignant tumors,and the mortality rate is second only to lung cancer.Because patients with early gastric cancer have no specific clinical symptoms and signs,most patients are diagnosed with advanced stage or even far away.Metastasis,poor treatment effect,at present,invasion and metastasis is still the most important cause of death of gastric cancer patients.Therefore,in-depth study of the molecular and regulatory mechanisms of gastric cancer invasion and metastasis is of great significance for improving the prognosis of gastric cancer patients.Epithelial Mesenchymal Transition(EMT)is an important biological process of tumor invasion and metastasis.The activation of epithelial mesenchymal transformation makes the tumor cells lose their adhesion,and it is easier to detach from the origin and migrate,and the tumor cells thus obtain a stronger invasion and metastatic ability.EMT plays an extremely important role in tumor invasion and metastasis.Previous studies have shown that E-cadherin combines with ?-catenin to form a complex that mediates cell polarity and stability of cell adhesion to regulate tumor invasion and metastasis.The degradation of P-catenin can induce cell EMT.TRIB3(Tribbles Homolog 3),is a member of the Tribbles homologous protein family and contains a serine/threonine kinase-like domain.The atypical pseudokinase domain provides a binding domain for substrates that can be recognized by the specific E3 ubiquitin ligase.In recent years,studies have shown that TRIB3 is highly expressed in a variety of tumor cells and participates in regulating the proliferation,migration and invasion of tumor cells.Further studies have confirmed that the high expression of TRIB3 is positively correlated with the poor prognosis of various tumors including lung cancer,breast cancer,and colon cancer.However,the biological role of TRIB3 in gastric cancer and its regulatory mechanism are not yet clear.Objective:Database analysis combined with a large number of gastric cancer in vivo and in vivo experiments to explore the role and clinical significance of TRIB3 in the invasion and metastasis of gastric cancer,explore the effect of TRIB3 on EMT process,and further clarify the mechanism of TRIB3 in the invasion and metastasis of gastric cancer,with a view to the invasion and metastasis of gastric cancer The molecular mechanism provides a new understanding,indicating whether the expression of TRIB3 can be used as a biological marker to evaluate the invasion and metastasis of gastric cancer.Methods:1.Database analysis of the expression of TRIB3 in gastric cancer and adjacent tissues,immunohistochemical detection of TRIB3 expression in clinical samples of gastric cancer and adjacent tissues,and analysis of the relationship between TRIB3 and the prognosis of gastric cancer patients.2.Transwell experiment and western blot experiment to analyze the relationship between the expression of various gastric cancer cell lines TRIB3 and invasion ability;3.To establish TRIB3 knockdown and overexpression gastric cancer cell lines through lentivirus infection,and to establish nude mice metastasis model,transwell invasion experiment and scratch experiment by tail vein injection to verify that TRIB3 promotes gastric cancer invasion and metastasis.4.Detect the expression of EMT-related markers and transcription factor ?-catenin after interfering with the expression of TRIB3 in gastric cancer cells by western blot,real-time quantitative PCR,etc.,clarify the effect of TRIB3 on EMT of gastric cancer cells,and explore TRIB3 by promoting the expression of ?-catenin In turn,it affects the regulation pathway of EMT.5.Add protein synthesis inhibitors and proteasome inhibitors to treat gastric cancer TRIB3 knockdown cells to determine whether TRIB3 affects its expression by affecting protein degradation of ?-catenin.6.To detect the effect of TRIB3 on the ubiquitination degradation of ?-catenin protein,co-immunoprecipitation to detect whether there is interaction between the two.7.Immunohistochemical staining to detect the expression of clinical tissue samplesTRIB3 and ?-catenin,analysis the correlation between TRIB3 and ?-catenin.Results:1.TRIB3 promotes malignant metastasis of gastric cancer(1)Database analysis.Among members of the Tribbles homologous protein family,only the expression of TRIB3 in gastric cancer is different from that in adjacent tissues,and the overall survival of patients with high expression of TRIB3 in gastric cancer is significantly lower than that of patients with low expression.Immunohistochemical staining also confirmed that the high expression of TRIB3 was significantly associated with poor prognosis,and the expression of TRIB3 in metastatic tissue was significantly higher than that of patients without metastasis.(2)Western blot results suggest that the expression of TRIB3 is positively correlated with the invasion ability of gastric cancer cells.The systemic metastasis model of tail vein injection in nude mice confirmed that after TRIB3 knockdown,the number of metastases on the lungs of nude mice was significantly lower than that of the control group.Transwell experiments and scratch experiments also confirmed that the invasion and metastasis ability of gastric cancer cell lines after TRIB3 knockdown was significantly reduced.The results of these in vivo and in vitro experiments confirmed that TRIB3 can promote the invasion and metastasis of gastric cancer.2.TRIB3 regulates the EMT process of gastric cancer cells through a post-transcriptional pathway(1)After TRIB3 knockdown,the expression of E-cadherin increased in EMT markers,while the expression of N-cadherin and Vimentin decreased,and the expression of key factor ?-catenin in Wnt/?-catenin signaling pathway decreased.(2)TRIB3 knockdown promotes ?-catenin protein expression,while TRIB3 knockdown does not affect ?-catenin mRNA expression.3.TRIB3 inhibits the ubiquitination degradation of ?-catenin and promotes EMT of gastric cancer cells.(1)The gastric cancer cell TRIB3 affects the degradation of ?-catenin protein through the ubiquitin proteasome system;(2)Knocking down TRIB3 promotes the ubiquitination degradation of ?-catenin;(3)There is mutual binding between TRIB3 and ?-catenin protein.(4)TRIB3 and ?-catenin protein expression were significantly positively correlated.Conclusion:Based on the above results,we draw the following conclusions:1.The expression of TRIB3 in gastric cancer tissues is higher than that in paracancerous tissues.In gastric cancer tissues,the expression of TRIB3 in metastatic patient tissues is significantly higher than that in patients without metastasis,and patients with high expression of TRIB3 have shorter survival times.2.TRIB3 promotes EMT of gastric cancer cells.This process is not achieved by regulating ?-catenin at the transcription level,but occurs in the post-transcriptional pathway.3.TRIB3 can inhibit the ubiquitination degradation of ?-catenin,make the abnormal accumulation of ?-catenin in the cell,and then promote the EMT of gastric cancer cells.There is mutual binding between TRIB3 and ?-catenin,and their protein expressions are related.4.TRIB3 can promote the invasion and metastasis of gastric cancer,can be used as a marker to evaluate the invasion and metastasis ability of gastric cancer,and may be one of the potential therapeutic targets for patients with gastric cancer metastasis.