The Mechanism Of TRAF7 Regulating Apoptosis And Epithelial-mesenchymal Transition In The Development Of Colon Cancer

BackgroundColorectal cancer(CRC)is one of the most common malignant tumors in the world,and incidence of CRC is on the rise.According to the latest statistics,the number of incidences and deaths of colorectal cancer in China ranks third and fifth respectively among all malignant tumors.Although the incidence rate of CRC has declined slightly in recent years,the economic burden of disease continues to rise.Epidemiological analysis shows that the risk of occurrence and death of colon cancer is higher than that of rectal cancer.Exploring the mechanism of colon cancer occurrence and development is of great significance for cancer prevention and control.Invasion and migration are important procedures in the development of colon cancer,which are closely related to the enhancement of cancer cell proliferation,reduction of apoptosis and epithelial-mesenchymal transition(EMT).JNK and NF-κB pathways are two of the common pathways that regulate tumor cell proliferation,apoptosis and EMT.Activation of JNK pathway can induce tumor cell apoptosis,while activation of NF-κB pathway promotes EMT,which is beneficial to tumor invasion and metastasis.Studies have reported that TRAF7 can regulate the activation of JNK and NF-κB pathways,and plays an important role in tumor cell apoptosis and EMT.At present,there are still few studies on the role and mechanism of TRAF7 in the development of colon cancer.ObjectiveTo explore the role and mechanism of TRAF7 regulating apoptosis and epithelial-mesenchymal transition in the development of colon cancer.MethodsThis research is divided into four parts:(1)The first part is a clinical retrospective study.We collected the medical history data,follow-up data and paraffin-embedded tissues of 115 patients with colon cancer,and used immunohistochemistry to detect the expression of TRAF7 in colon cancer tissues and normal colon mucosa tissues.Then analyze the impact of TRAF7 on the prognosis of colon cancer patients through the cox regression model.(2)In the second part,HT-29 colon cancer cells with empty plasmid(si-Control,OE-Control),Traf7 overexpression(OE-TRAF7)and Traf7interference(si-TRAF7)were obtained by plasmid transfection technology.After stimulating the cells with TNF-α,the apoptosis and proliferation levels of colon cancer cells with high and low expression of TRAF7 were compared using flow cytometry apoptosis detection,MTT and cell cloning test.The expression levels of JNK,p-JNK,C-FLIP,Caspase-9 and Caspase-3 were detected by Western blot,and the effect of TRAF7 on the activation of JNK pathway in colon cancer cells was analyzed.(3)In the third part,the migration ability of untransfected HT-29 cell line(Control),si-Control,si-TRAF7,OE-Control and OE-TRAF7 cell lines were detected by scratch test,exploring the influence of TRAF7 expression on the migration ability of colon cancer.Then detect the expression of E-cadherin,N-cadherin,Snail,Slug,ZEB1,ZEB2 and TWIST1 by PCR and Western blot,and analyze the effect of TRAF7 on colon cancer cell EMT.Finally,Western blot was used to detect the expression levels of IκBα,p-IκBα,p65 and p-p65,and analyze the effect of TRAF7 on NF-κB activation.(4)The fourth part uses the human-derived tumor cell xenotransplantation technology to inoculate the sh-Control,sh-TRAF7,OE-Control,and OE-TRAF7HT-29 cell line into BALB-c/nu mice subcutaneously.The volume and the weight of the subcutaneous tumor was measured after 20 days,and the effect of TRAF7 on the growth,NF-κB and JNK was analyzed.Results(1)The expression of TRAF7 in colon cancer tissue was lower than that in normal colon mucosa tissue(P<0.05).The univariate cox regression model showed that tumor stage,lymphatic metastasis,distant metastasis,degree of surgical resection,surgical method and TRAF7 expression all have an impact on the prognosis of colon cancer patients(all P<0.05).After correction using the multivariate cox model,it was found that low expression of TRAF7 can reduce the survival time of colon cancer patients(P<0.05).(2)The proliferation activity,clone formation rate and C-FLIP expression of OE-TRAF7 cell line decreased,the expression of p-JNK,Caspase-9,Caspase-3 increased(all P<0.05).While the si-TRAF7 cell line showed the opposite trend(all P<0.05).(3)The cell migration rate of OE-TRAF7 cell line decreased,the expression of E-cadherin increased,and the expression of Snail,Slug,ZEB1,ZEB2,TWIST1,N-cadherin,IκBα,p-IκBα,p65 and p-p65 decreased(all P<0.05).While the si-TRAF7 cell line showed the opposite trend(all P<0.05).(4)20 days after transplantation,the subcutaneous tumors of nude mice in the OE-TRAF7 group were smaller than those in the OE-Control group,and the expression of p-JNK increased,and the expressions of IκBα,p-IκBα,p65 and p-p65 decreased(all P<0.05),while the sh-TRAF7 group mice are opposite(all P<0.05).ConclusionsThe high expression of TRAF7 is beneficial to the prognosis of colon cancer patients.TRAF7 can promote the apoptosis of colon cancer cells and inhibit their proliferation activity by activating the JNK pathway.In addition,up-regulation of TRAF7 can inhibit the NF-κB pathway,reduce the occurrence of EMT of colon cancer cells,and weaken the migration ability of colon cancer cells.