Biological Effect And Underlying Mechanism Of Endoplasmic Reticulum Oxidoreductin Protein ERO1L In The Pancreatic Cancer

Pancreatic cancer is one of the most common malignancies with very high levels of malignancy in the digestive tract.In recent years,the incidence of pancreatic cancer in China is high,and the incidence of pancreatic cancer in the eastern and northeastern regions has exceeded the average in the traditional high-risk countries in the West.At present,surgical resection is still considered to be the best clinical treatment for PDAC.However,only 10-20%of new pancreatic cancer patients are evaluated for surgery after evaluation.The effect of pancreatic cancer treatment is poor,the mortality and morbidity rate are similar,the 5-year survival rate is extremely low,and the prognosis is extremely poor.It is urgent to explore the mechanism of occurrence and development of pancreatic cancer and new treatment methods.Endoplasmic reticulum oxidoreductase 1 alpha(ERO1L)is located in the endoplasmic reticulum(ER).It is an enzyme molecule containing flavin adenine nucleotides and is involved in the formation of disulfide bonds.ERO1L effectively re-oxidizes prolyl 4-hydroxylase beta/protein disulfide isomerase(P4HB/PDI),accepts the electrons of the reduced PDI and transmits them to the molecule Oxygen catalyzes the formation of disulfide bonds between secreted proteins and cell surface proteins,and also leads to the production of reactive oxygen species(ROS)in cells.This is a necessary condition for the normal folding of immunoglobulins.Studies have confirmed that ERO1L is highly expressed in a variety of malignant tumors,such as lung cancer,gastric cancer,and breast cancer.It plays an important role in tumor growth,metastasis,angiogenesis and immune escape,and is closely related to the prognosis of tumor patients.However,the current research on the expression of ERO1L in pancreatic ductal adenocarcinoma is still very limited,and its mechanism of action needs to be further explored.This project first screened the pancreatic cancer-related gene ERO1L through whole exome and transcriptome sequencing of liver metastatic pancreatic cancer combined with public database data.And further study the expression of ERO1L in pancreatic cancer and its correlation with prognosis,verify the effect of ERO1L on the biological function of pancreatic cancer cells,and finally clarify the possible molecular mechanism of ERO1L regulating the occurrence,development and metastasis of pancreatic cancer.This dissertation consists of the following four parts:Part Ⅰ Pancreatic cancer liver metastasis whole exome and transcriptome sequencing to screen pancreatic cancer-related genes ERO1LObjective:To screen pancreatic cancer liver metastasis-related genes using whole exome and transcriptome sequencing technology.Methods:Synchronous surgically resected samples from 17 hepatic oligometastatic pancreatic ductal adenocarcinoma patients were enrolled in this study.Thirty-one paraffin tissue samples were used to extract DNA and perform whole-exome sequencing.RNA was extracted from 33 fresh tissue samples for transcriptome sequencing.Bioinformatics analysis of sequencing data.Results:The genomic mutations of the primary pancreatic cancer and liver metastases were highly consistent;genes related to cell stemness were highly expressed in samples from the disease environment;pathways that were up-regulated in tumor tissues showed a variety of different trends;inflammation and immune response may play an important role in liver metastasis of pancreatic cancer.Conclusion:The transcriptome heterogeneity of the primary pancreatic cancer and metastasis in the same patient is small.In the process of tumorigenesis,tumor tissues and surrounding non-tumor tissues change together;in the process of disease progression,tumor cells create favorable conditions for themselves through changes in their own transcriptome and the connection between them and surrounding tissues.Immune-related genes are highly expressed in pancreatic cancer,providing new possibilities for the treatment of pancreatic cancer.Part Ⅱ The expression of ERO1L in pancreatic cancer and its correlation with prognosisObjective:To study the expression of ERO1L in pancreatic cancer tissues and its correlation with the prognosis of pancreatic cancer.Methods:205 cases of pancreatic ductal adenocarcinoma tissues and paired normal pancreatic tissues were enrolled in the study;Analyze the expression data of normal tissues and tumor tissues including pancreatic cancer from the Oncomine,TCGA,GTEx public databases.Results:The expression level of ERO1L is different basing on tumor type,and it is highly expressed in a variety of tumors including but not limited to cervical squamous cell carcinoma,cervical adenocarcinoma,lung adenocarcinoma,and pancreatic ductal adenocarcinoma.Kaplan-Meier survival analysis shows that the expression pattern of ERO1L is related to the prognosis of patients.Inversely,the overall survival rate of patients with pancreatic ductal adenocarcinoma(PDAC)with higher ERO1L expression was shorter than that of patients with lower ERO1L expression(P=0.0078).By immunohistochemical staining,it was found that compared with normal pancreatic tissue,human pancreatic intraepithelial neoplasia(PanIN)and PD AC tissues increased the expression level of ERO1L protein.Tumor size(P=0.002)and histological differentiation(P=0.033)were significantly different between the high ERO1L expression group and the low ERO1L expression group.Conclusion:ERO1L expression is up-regulated in PD AC tissue,and its expression is significantly related to the patient’s tumor size and histological differentiation.ERO1L is a predictor of poor prognosis of pancreatic cancer.The higher the level of ERO1L expression in pancreatic cancer tissue predicts the worse the prognosis.Part Ⅲ The effect of ERO1L on the biological functions of pancreatic cancer cellsObjective:To study the effects of overexpression and knockdown of ERO1L expression in different human pancreatic cancer cell lines using ERO1L inhibitors on the proliferation,migration and invasion of pancreatic cancer cells.Methods:Constructing a stably transfected ERO1L interfering pancreatic cancer cell line and using ERO1L inhibitor,using CCK8,plate cloning experiment and other methods to detect the changes in the proliferation ability of pancreatic cancer cells after treatment.The transwell chamber was used to detect the changes in the invasion and migration ability of pancreatic cancer cells.Animal experiments tested the effect of inhibitor treatment on tumor size and liver metastasis in mice.Results:ERO1L was highly expressed in different human pancreatic cancer cell lines.Short hairpin RNA(shRNA)was used in two PDAC cell lines(Capan-2 and MiaPaCa-2)with higher ERO1L protein levels)Silencing ERO1L,the expression level of ERO1L was significantly decreased in these two cell lines(>80%),and the migration and invasion ability of PD AC cells in these two cell lines was also inhibited.In addition,in vitro cell experiments and animal experiments have shown that pharmacologically inhibiting the expression of ERO1L can significantly limit the growth,migration,invasion and other tumorigenesis and development processes of PDAC cells.Conclusion:ERO1L is highly expressed in different human pancreatic cancer cell lines.Inhibiting the expression of ERO1L can limit the growth,migration and invasion of PDAC cells,providing a new potential target for the treatment of pancreatic cancer.Part Ⅳ ERO1L regulates the molecular mechanism of pancreatic cancerObjective:To explore the potential molecular mechanism of ERO1L in regulating the proliferation,invasion and migration of pancreatic cancerMethods:Bioinformatics analysis explores the possible mechanism of ERO1L’s role in pancreatic cancer;ER-stress inhibitor treats pancreatic cancer cell lines to study its effect on ERO1L expression;Seahorse energy analyzer is used to detect cellular oxygen consumption rate(OCR)and Extracellular acidification rate(ECAR),using a kit to detect the consumption of glucose and lactic acid production in the culture supernatant to study the effect of ERO1L on aerobic glycolysis of pancreatic cancer.Results:Treatment of pancreatic cancer cells with ER-stress chemical inducer showed that it can activate the expression of ERO1L and ER-stress-related proteins.TCGA data analysis found that ERO1L expression is related to UPR,hypoxia and glycolysis pathways.Glucose uptake experiments found that interference with ERO1L reduced the sugar uptake of pancreatic cancer cells.Interfering with EROIL expression inhibits aerobic glycolysis and promotes oxidative phosphorylation.Overexpression of EROIL promoted aerobic glycolysis of pancreatic cancer cells and inhibited its oxidative phosphorylation.Compared with glucose,galactose enters aerobic glycolysis with lower efficiency.After high expression of EROIL,aerobic glycolysis significantly enhances the proliferation and growth of pancreatic cancer cells.The use of glycolysis inhibitors 2-FDG and 2-DG significantly inhibits the promotion of ERO1L.Proliferation and growth.GSEA analysis showed that ERO1L may promote pancreatic cancer metastasis through EMT.Further cell experiments showed that E-cardherin was up-regulated and Vimentin was down-regulated after interfering with ERO1L or using inhibitors,inhibiting the EMT pathway.Conclusion:ER-stress induces the expression of ERO1L in pancreatic cancer cells,promotes the level of aerobic glycolysis of pancreatic cancer cells and promotes the proliferation and growth of pancreatic cancer cells by activating aerobic glycolysis.In addition,ERO1L promotes liver metastasis of pancreatic cancer by activating EMT.