The Mechanism Of RUNX1 In Gemcitabine-Resistance Of Pancreatic Cancer Through Endoplasmic Reticulum Stress

Objective:RUNX1 is a member of the RUNX family which participates in the regulation of cell lineage-specific genes,cell differentiation and development.Early studies mainly focus on hematological diseases.More and more studies have found that RUNX1 plays a role in promoting malignant progression or inhibiting proliferation of tumors in various solid tumors.RUNX1 can promote epithelial-mesenchymal transformation(EMT)in pancreatic cancer and enhances tumor invasion and migration.However,the relationship between RUNX1 and chemotherapy resistance in pancreatic cancer has not been reported.In the previous analysis of TCGA data,we found that RUNX1 expression may be associated with the therapeutic efficacy of pancreatic cancer.Therefore,this study was designed to clarify the relationship between RUNX1 and gemcitabine resistance in pancreatic cancer,explore the molecular mechanism,and evaluate the potential value of RUNX1 in pancreatic cancer and the predictor of chemotherapeutic efficacy.Methods:Immunohistochemistry was used to detect the level of related proteins in tissues;CCK8 was used to detect cell viability;flow cytometry was used to detect apoptosis;real-time fluorescent quantitative PCR was used to detect mRNA levels of related genes;Western blot was used to detect protein levels of related indicators;transmission electron microscopic observation Endoplasmic reticulum structure;liposome transfection to establish transient expression or knockdown cell lines;molecular cloning and viral infection to construct stable expression cell line;chromatin immunoprecipitation technique(ChIP)to detect protein-DNA binding;the dual luciferase reporter gene system detects the transcriptional activity of the protein after binding to the DNA sequence;the subcutaneous tumor formation test was used to observe the effect on the tumor growth and evaluate the drug response.Results:1.Clinical data analysis showed that RUNX1 expression predicts poor prognosis of pancreatic cancer.Patients in the RUNX1 high expression group had a lower T stage,a lower degree of tissue differentiation,and a higher incidence of lymph node metastasis.Kaplan-Meier survival analysis showed that the RUNX1 low expression group had a significantly longer survival.2.TCGA database analysis suggested that RUNX1 expression was associated with late treatment of pancreatic cancer patients.The expression of RUNX1 in the completed remission group of pancreatic cancer patients was significantly lower than that in the disease progression group,and the median survival time of the high RUNX1 expression in the disease progression group was shorter.In vitro,the cell viability of RUNX1 overexpressing pancreatic cancer cells treated with gemcitabine was enhanced compared with the control group,and the proportion of apoptosis was decreased.Conversely,when the expression of RUNX1 decreased,the survival rate of pancreatic cancer cells treated with gemcitabine decreased significantly,and the proportion of apoptosis increased.Similarly,when the expression of RUNX1 was down-regulated in gemcitabine-resistant cell lines,cytotoxicity tests suggested that decreased expression of RUNX1 reversed the sensitivity of drug-resistant strains to gemcitabine.3.Under the electron microscope,24 hours after the action of gemcitabine in pancreatic cancer cells,a large number of vesicular-like changes occurred in the normal squamous cytoplasmic reticulum structure in the cells,which were irregularly arranged and consistent with changes after endoplasmic reticulum stress.The blocking experiments in the RUNX1 overexpression group showed an increase of apoptosis rate induced by gemcitabine after blocking endoplasmic reticulum stress.The phosphorylation levels of BiP and UPR pathways such as eIF2 a protein increased with the up-regulation of RUNX1 expression,and decreased with the down-regulation of RUNX1 expression.4.TCGA database analysis showed a positive correlation between RUNX1 and HSPA5 expression(R=0.32,P<0.001).Real-time quantitative PCR and Western blot were used to detect positive correlation between the expression level of BiP and RUNX1.Chromatin immunoprecipitation(ChIP)assay detects that RUNX1 binds to the corresponding sequence of the BiP promoter region.The dual luciferase assay showed that the transcriptional activity of BiP was stronger in the overexpressing RUNX1 group than in the control group.5.In the subcutaneous tumor model of pancreatic cancer in nude mice,the tumor volume and tumor weight of the gemcitabine group,the Ro5-3335 group and the combination group were significantly different from those of the control group.The weight loss was obvious in the group treated with gemcitabine alone or combination with Ro5-3335.In addition,when the expression of RUNX1 was down-regulated,the tumor growth rate was significantly slowed down,and the inhibitory effect of gemcitabine on the tumor was more obvious.The tumor growth rate was significantly accelerated after the restoration of RUNX1 expression.Conclusions:1.RUNX1 can be used as a predictor of poor prognosis in patients with pancreatic ductal adenocarcinoma.2.RUNX1 reduces the sensitivity of pancreatic cancer cells to gemcitabine.3.RUNX1 enhances the endoplasmic reticulum stress regulation system and leads to a decrease in the sensitivity of cells to gemcitabine through the endoplasmic reticulum stress pathway.4.RUNX1 directly activates BiP transcription,which up-regulates BiP expression and promotes endoplasmic reticulum stress response.5.RUNX1 inhibitors have the effect of inhibiting pancreatic cancer that is are more toleranted.RUNX1 inhibitor combined with gemcitabine alone can increase tumor inhibition.