Mechanism Of SOX8 Affecting The Sensitivity Of Albumin-Bound Paclitaxel In Pancreatic Cancer

BackgroundPancreatic cancer is one of several digestive tract tumors with extremely poor prognosis.In recent years,its incidence and mortality have been increasing year by year in the world including China.Pancreatic cancer is a highly invasive tumor,which is usually characterized by high metastasis and incomplete resection.The resection rate is less than 20% and the postoperative recurrence rate is high.Its main treatments include surgery,chemotherapy and radiation.Although new advances have been made in surgical techniques,chemotherapy and radiotherapy regimens over the years,due to low operability and poor sensitivity to chemoradiation,it is of vital importance to select highly sensitive chemotherapy drugs in treatment.SOX8 is a member of the sox gene family,which encodes a transcription regulator,and plays an extremely important role in determining sex and embryo development.More and more studies have shown that abnormal expression of SOX8 gene is closely related to the occurrence and development of tumors.Therefore,by exploring the expression changes of SOX8 in pancreatic ductal adenocarcinoma and its significance in the treatment of pancreatic cancer,we designed this research.MethodParaffin samples of tumor tissues with definite pathological diagnosis after clinical treatment of pancreatic duct adenocarcinoma were collected.SOX8 antibodies of three different companies with different clone numbers were selected for immunohistochemical staining analysis to determine the distribution and expression of SOX8 protein in pancreatic duct adenocarcinoma tumor tissues,and statistical treatment was performed.Combined with relevant clinical data,the relationship between SOX8 expression level and the clinical indicators,pathological indicators and different reactions after drug treatment in the above pancreatic cancer patients was analyzed.Furthermore,the sensitivity of the above patients to receive chemotherapy with the albumin-bound paclitaxel regimen was further analyzed,and regular review and follow-up were conducted for further survival analysis.To construct SOX8 overexpression and down-expression stable pancreatic cancer cell lines,as well as SOX8 overexpression plasmid and interfering RNA.Stability and transient transfection results were verified at protein and mRNA levels.Observe different SOX8 level expression of pancreatic ducts in albumin gland cancer cells combined with the reaction of different type of taxol drug tolerance,respectively using western blot and immunofluorescence observed expression,downexpression or interference of different cells after SOX8 tubulin depolymerization changes,using flow cytometry to further analyze the changes of the cell cycle,according to the change of the cell cycle,observing the relationship between ?-catenin and SOX8 by western.The relationship between them is further analyzed with TCGA database.According to relevant research results and TCGA database analysis,there is a positive regulating relationship between EZH2 and ?-catenin.By applying the promoter region binding array analysis,EZH2 accept the transcriptional regulation of SOX8,and the expression level of EZH2 and ?-catenin is verified by Western blot.Chromatin Immunoprecipitation Assay(ChIP,Chromatin Immunoprecipitation Assay)and double luciferase were used to further clarify the role of SOX8 in direct transcriptional regulation of EZH2 in pancreatic cancer cells.Respectively by nuclear plasma separation experiments extraction pancreatic cancer cell nucleus and cytoplasm protein,Western Blot detection after SOX8 overexpression and downexpression the change of EZH2,beta-catenin,and observed by immunofluorescence experiments SOX8 overexpression and downexpression in pancreatic cancer cell line accept albumin,combined type paclitaxel treatment after the change of cell microtubule protein,further verify whether there is a correlation between the indexes.Finally,the regulatory relationships between SOX8,EZH2,and ?-catenin were further determined through the corresponding blocking and reply experiment,and the possible mechanism of the sensitivity of SOX8 reactive pancreatic cancer cell lines to the treatment of reactive albumin-bound paclitaxel was studied.The subcutaneous tumorigenesis of pancreatic carcinoma was established in nude mice with different expression of SOX8,and the relationship of SOX8 to the drug response after the treatment with albumin-bound taxol and EZH2 inhibitor was evaluated by intraperitoneal injection.ResultsExpression of SOX8 in tumor tissues and adjacent tissues of ductal adenocarcinoma and its related clinical significance.Immunohistochemical staining was performed on the surgical specimens of 100 cases of pancreatic ductal adenocarcinoma,and it was found that there were some differences in the expression of SOX8 in tumor tissues.According to the expression of SOX8 in immunohistochemical tests,the corresponding clinical data were divided into two subgroups,the SOX8 high-expression group and the SOX8 low-expression group.Kaplan-meier analysis revealed that the prognosis of patients with increased SOX8 was significantly worse(DFS was 12.6 months vs.6.6 months,P<0.05).This data is consistent with TCGA database results.The clinical data of 100 patients undergoing surgical treatment for pancreatic ductal adenocarcinoma were further analyzed,with high expression of SOX8 accounting for 69.2%(81 cases),and low expression of SOX8 accounting for 30.8%(36 cases).The correlation between SOX8 protein expression level and pathological parameters was analyzed through clinical data.It was found that patients with high SOX8 expression in pathological sections of pancreatic cancer were more likely to have lymph node metastasis than those with low SOX8 expression.The expression level of SOX8 protein in pancreatic ductal adenocarcinoma cells was related to apoptosis and cell cycle changes after treatment with albumin-bound paclitaxel.According to the basic expression levels of SOX8 in pancreatic cancer cell lines,lentiviral infection was used to successfully construct the pancreatic ductal adenocarcinoma cell lines(cf-pac1 and PANC-1)with overexpression of SOX8 and the cell lines with downexpression of SOX8(CFPAC-1 and MIA PaCa-2).Compared with the low expression in pancreatic cancer cells,the high expression of SOX8 in pancreatic cancer cells were treated with the albumin paclitaxel,the apoptosis and the microtubulin aggregation were reduced.The expression level of SOX8 protein in pancreatic ductal adenocarcinoma cells was significantly positively correlated with the expression level of ?-catenin(R=0.301,**p<0.01).SOX8 overexpression of pancreatic adenocarcinoma ?-catenin and its downstream cycle-related proteins: CyclinD1(CyclinD1)and c-myc were increased compared with the wild control group.And successive in pancreatic cancer tissue paraffin section of positioning experiment confirmed SOX8 consistency with ?-catenin expression,flow cytometry tests verify SOX8 lower expression of pancreatic cancer cells to accept type albumin in combination with paclitaxel treatment after more gathered in G2,albumin and SOX8 expression form of pancreatic cancer cells to accept type combination paclitaxel treated agglomeration of G2 in relatively SOX8 lower expression of pancreatic cancer cells was reduced.After upregulation of ?-catenin in the cell lines with low expression of SOX8,it was found that the concentration of G2 stage was reduced in the pancreatic cancer cells with lower expression of SOX8.As a transcription regulator,SOX8 is directly involved in the expression of EZH2 in pancreatic ductal adenocarcinoma cells.The protein and mRNA levels of SOX8 and EZH2 in different stable cell lines were detected by Western blot and realtime quantitative PCR.The results showed that the expression level of EZH2 in pancreatic duct cancer cells was directly regulated by SOX8,showing a significant positive correlation.Bioinformatics analysis suggested that there exists potential sequence of SOX8 transcription regulation and binding in the EZH2 promoter region.By using CHIP(chromatin immunoprecipitation)technology,the binding site of transcription regulator SOX8 and EZH2 promoter region was confirmed.Thus construct EZH2 promoter regions report gene carrier,dual luciferase reporter gene SOX8 direct combination is verified by the experiments and the expression of transcription activation EZH2,mutate the above SOX8 in EZH2 promoter regions binding sites,transcription activity was significantly suppressed,confirmed SOX8 gland cancer cells can directly regulate the expression of EZH2 and regulate the expression of ?-catenin.Primitive cells build people pancreatic tumor of nude mice subcutaneously into pancreatic cancer tumor model,by intraperitoneal injection of albumin in combination with paclitaxel and EZH2 inhibitor,results suggest SOX8 high expression of primitive cells of pancreatic tumors subcutaneously into pancreatic cancer tumor in nude mice tumor after application and albumin in combination with paclitaxel therapy to reduce the SOX8 low expression group the tumor inhibitory effect.The effect of EZH2 inhibitor on tumor inhibition was improved.Conclusion1.There were differences in the expression of SOX8 protein in pancreatic duct adenocarcinoma tissues,and the high expression of SOX8 suggested poor prognosis in pancreatic duct adenocarcinoma patients.2.SOX8 beta catenin expression and tumor cells ?-catenin and its downstream of the cell cycle related proteins: cell cycle protein D1(CyclinD1),C-myc expression levels showed positive correlation,the relationship between SOX8 lower expres sion of pancreatic cancer cells to accept type albumin in combination with paclitaxel treatment after more gathered in G2,Increased SOX8 indicates a decrease in the response of the corresponding tumor cells to albumin-binding paclitaxel.3.The expression of SOX8 is positively correlated with the expression level of EZH2 protein in tumor cells.The promoter region of EZH2 has the target sequence of SOX8 transcription regulation and binding.For pancreatic cancer cells,SOX8 can directly bind to the specific target sequence in the promoter region of EZH2,activate the transcription of EZH2,and further up-regulate the expression of EZH2 and downstream ?-catenin.4.Drug therapy in a nude mouse model of subcutaneous tumorigenesis in human pancreatic cancer after the construction of a human pancreatic cancer model suggested that the inhibition effect of SOX8 low expression group on the albuminbound paclitaxel was weakened.The effect of EZH2 inhibitor on tumor inhibition was improved.SOX8 can predict the drug treatment effect of albumin-bound paclitaxel in pancreatic cancer patients,and is expected to be a screening marker for the treatment effect of albumin-bound paclitaxel.