The Mechanism Of HMGB1-RAGE Inflammatory Signaling Pathway In Promoting The Progression Of Rectal Cancer

Objective:In recent years,more and more evidence shows that HMGB-1 and RAGE are not only related to the inflammatory diseases,but also closely associated with the incidence of a variety of tumors.Studies have proven that their expression increases significantly in many tumor tissues and which increases the invasiveness of the tumors,and may be related to a number of tumor signaling pathways including the K-RAS pathway,however,their role in rectal cancer and its specific mechanism remain unclear.Therefore,in this study,we will explore the alteration expression levels of HMGB-1 and RAGE and the roles and mechanisms of HMGB-1 and RAGE in the progressed rectal cancer cells and tissues,and forward to provide a scientific basis for the therapeutic target of the rectal cancer.Methods:1.In vitro culture of HCT116 and SW480 rectal cancer cells,HMGB-1 and RAS inhibitors named trans farinyl thiosicicylic acid(FTS)were added,and the proliferation of the colorectal cancer cells was detected by CCK-8.2.In vitro culture of HCT116 rectal cancer cells,the observation group added HMGB-1 for treatment,extraction of cell protein,K-RAS antibodies were used for immunoprecipitation and elution.Then RAGE and K-RAS antibodies were added to the cells,and labeled with second antibody The expression,interaction and changes of RAGE and K-RAS were analyzed by Western blot.3.In vitro culture of HCT116 rectal cancer cells,the observation group added HMGB-1 for treatment,adding RAGE,K-RAS antibody,and two anti markers,and double immunofluorescence double labeling analysis of RAGE,K-RAS expression,interaction and change.4.In vitro culture of HCT116 and SW480 rectal cancer cells and divided into 6 groups,group A:HCT116 cells;group B:HCT116 cell+HMGB1(100ng/ml)treatment group;group C:HCT116 cell+HMGB1(100ng/ml)treatment+RAS inhibitor treatment group;Group D:SW480 cells;Group E:SW480 cells + HMGB1(100ng/ml)treatment group;Group F:SW480 cells+HMGB1(100ng/ml)treatment + RAS inhibitor treatment group.Extract the total RNA of cells in above groups respectively,design and synthesize primers used by reverse transcriptase PCR with the Primer Premier 5 software of Shanghai raw Industry Corporation,use the housekeeper gene GAPDH as the internal parameter,RT-PCR to detect the expression of the target gene Yap1 expression in each cell.5.In HCT116 cells,HCT116+HMGB1 treated group cells and+RAS inhibitor cells in HCT116+HMGB1 treatment group,Western blot method was used to detect the protein level of the target protein Yap1.6.8 pairs biopsies of rectal cancer and paracancer from the Hospital Affiliated to Nantong University were selected.All patients were not treated with chemotherapy or radiotherapy before the operation,and all patients were suffered from no other inflammatory or tumor disease.Western blot was used to detect the expression of RAGE and Yap proteins.7.A total of 66 specimens of the paraffin section of the Hospital Affiliated to Nantong University were selected and all the patients were not treated with chemotherapy or radiotherapy before the operation,and all patients were excluded other inflammatory or tumor disease.The expression of RAGE and Yapl was detected by immunohistochemical method.The results of immunohistochemistry were judged by double blind method.The results of the test made the 2 senior Pathologists study respectively.Statistical method was used to analyze the relationship between the test results and pathological indicators.8.The tissues of rectal cancer were selected,the tissue protein was extracted,Immunocoprecipitation with K-RAS antibody was carried out,then elution was carried out,and then RAGE and K-RAS antibodies were added and labeled with second antibody,at last the expression and interaction of RAGE and K-RAS in rectal cancer tissues were analyzed by Western blot.Results:1.HMGB1 can significantly enhance the proliferation of HCT116 and SW480 rectal cancer cells,while RAS inhibitor FTS combined treatment can reduce the proliferation of HMGB1-mediated rectal cancer cells.2.Immunoprecipitation experiment was carried out in the rectal cancer cells after grouping with K-RAS antibody.The experimental results showed that RAGE and K-RAS in group A(HCT116 cell group)and B group(HCT116 cell+HMGB1 group)played an important role,and the function of B group was more obvious than that of A group.3.The results of double immunofluorescence test showed that RAGE and K-RAS were Co located in group A(HCT116 cell group)and group B(HCT116 cell+HMGB1 group).Compared with group A,the expression of K-RAS and RAGE increased significantly after adding HMGB1.4.In HCT166 and SW480 cells,the results of RT-PCR detection showed that the expression level of Yapl in the target gene increased significantly after the addition of HMGB1,and the expression level of the target gene was significantly decreased when RAS inhibitor was added on this basis,but it was still higher than the control cell group.5.In HCT166 cells,the results of Westen blot detection showed that the expression level of the target protein Yap1 was significantly increased after the addition of HMGB1,and the expression level of the target protein decreased significantly after adding RAS inhibitor on this basis,but it was still higher than the control cell group.6.The results of Westen blot detection in rectal cancer tissues and para cancerous tissues showed that the expression of two proteins of RAGE and Yapl in cancer tissues were higher than those in the para cancerous tissues in their respective samples.7.Immunohistochemistry showed that RAGE and YAP1 were overexpressed in rectal cancer specimens.The high expression of RAGE and YAP1 was related to the advanced histological grade,lymph node metastasis and TNM staging.8.Immunoprecipitation was performed with K-RAS antibody in rectal cancer tissue.The results showed that both RAGE and K-RAS were highly expressed in rectal cancer tissues,and the interaction between the two proteins was found.Conclusions:1.HMGB1-RAGE signalling can promote the proliferation of rectal cancer cells,and the addition of RAS inhibitors can weaken the proliferation of rectal cancer cells mediated by HMGB1-RAGE signal.RAGE and K-RAS interact with each other in the progression of rectal cancer.HMGB1 can enhance their expression and interaction.2.In rectal cancer cells,HMGB1 could enhance the expression of Yapl.When the RAS inhibitor was added on this basis,the expression level of Yapl was significantly decreased.The high expression of RAGE and YAP in rectal cancer is related to the invasiveness of the tumor,and there is a positive correlation between them.3.Chronic inflammation up-regulates the expression of HMGB1-RAGE,promotes the activation of RAGE and the recruitment of K-RAS,activates Yap 1 protein downstream of RAS,and promotes the progression of rectal cancer.