The Role And Mechanisms Of Transcription Factor NRF2 In The Carcinogenesis And Malignant Behaviors Of Pancreatic Cancer

Objective:In recent years,the incidence of pancreatic cancer has been increasing worldwide.According to the latest statistics from China’s National Cancer Center,pancreatic cancer ranks eighth in the incidence of malignant tumors in urban chinese men and sixth in the mortality rate of malignant tumors in large cities.Due to the characteristics of insidious onset,early metastasis and poor therapeutic effect of pancreatic cancer,patients were in the middle and late stage when they were diagnosis and lost the opportunity of radical surgical treatment.In order to improve the prognosis of patients with pancreatic cancer,we need to conduct more in-depth studies on the pathogenesis and development of pancreatic cancer.The occurrence and development of tumor are closely related to the imbalance of intracellular redox homeostasis.Nuclear factor erythroid 2-related factor 2（NRF2/NFE2L2）plays a particularly important role of intracellular redox homeostasis regulation by regulating transcription and translation of downstream antioxidant genes.More and more literature reports have confirmed that NRF2 mediates different effects in different types of tumors,even in different stages of the same tumor,that is,it has a bidirectional regulation effect on the occurrence and development of tumors.At present,there are no specific in vivo studies on the key role of NRF2 in the development of pancreatic cancer,and there are few reports on the specificity of NRF2 in human pancreatic cancer epithelial-mesenchymal transformation and other malignant biological behaviors.In conclusion,this study used pancreatic tissue-specific Nrf2 knockout Kras mutant mouse model and NRF2-silenced pancreatic cancer cell line to explore the role and mechanism of NRF2 in the occurrence and malignant biological behavior of pancreatic cancer,providing a new theoretical basis for the prevention and treatment of pancreatic cancer.Methods:1.NRF2 transcriptome data set and related clinical data set were downloaded from TCGA public database to analyze the differential expression of NRF2 in pancreatic cancer tissues and normal tissues and the correlation between NRF2 expression level and general clinical data,pathological data and prognosis of patients.Immunohistochemical staining,q RT-PCR and Western Blot were used to detect the expression of NRF2 and its downstream target genes in 12 pairs of pancreatic cancer tissues and corresponding adjacent tissues collected in our department.Meanwhile,serum CA19-9 level indexes were collected at admission to analyze the correlation between the two expression levels.The clinical correlation between differential expression and patients was further verified.2.Nrf2^Loxp/Loxpand Pdx1-Cre mice were used to construct CN(Nrf2^Loxp/Loxp;Pdx1-Cre)mice were used to achieve pancreatic tissue-specific Nrf2 knockout,and the effects of Nrf2deletion on pancreatic development and pancreatic secretory and exocrine functions were observed.Meanwhile,KC mice with pancreatic tissue-specific Kras mutation(LSL-Kras^G12D/+;Pdx1-Cre)and constructed pancreatic tissue-specific Nrf2 knockout Kras mutant mice KCN mice((LSL-Kras^G12D/+;Nrf2^Loxp/Loxp;Pdx1-Cre).It aims to investigate the role of Nrf2 in the pathogenesis of pancreatic cancer.Body weight,diet and water intake were monitored weekly.Blood and pancreatic tissue samples were collected at 4,8and 16 weeks,respectively,and serum amylase activity was detected also in these times.Hematoxylin-eosin staining（HE staining）and immunohistochemical staining（CK-19,NQO-1,Ki-67,8-OHd G）were performed on pancreatic tissues after fixation with 4%paraformaldehyde.Total RNA was extracted from pancreatic tissue to detect the transcription level of genes related to oxidative stress and interstitial response.Total protein of pancreatic tissue was extracted to detect NRF2 and its downstream target genes as well as key proteins and signaling pathways in epithelial cell proliferation response.3.The expression levels of NRF2 and its downstream target genes in three pancreatic cancer cell lines（As PC-1,CAPAN2,PANC-1）and normal pancreatic duct cells（HPDE6-C7）were detected by q RT-PCR and Western Blot.In addition,inhibition or overexpression of NRF2 in CAPAN2 and PANC-1 cells were achieved by lentivirus transfection technique.Subsequently,cell counting,Ed U assay,monoclonal formation assay,cell scratch assay and Transwell assay were used to detect the changes in proliferation,migration and invasion of pancreatic cancer cells after overexpression and inhibition of NRF2 expression.The correlation between the expression level of NRF2 and EMT and glycolysis-related proteins or transcription factors was detected by second-generation sequencing technology,and the differential genes were analyzed by pathway enrichment.These genes were also verified by WB and q RT-PCR,thus to explore the mechanism of NRF2 in pancreatic malignant biological behavior.Results:1.The expression level of NRF2 in pancreatic cancer tissues was significantly higher than that in adjacent tissues,and was significantly correlated with the clinicopathological grade and CA19-9 level of patients,which could be used as a risk factor for prognosis of patients.TCGA database analysis showed that the expression level of NRF2 in pancreatic cancer tissues was significantly higher than that in normal tissues.The analysis of 12 pairs of clinical pancreatic cancer tissue pathology samples also confirmed that NRF2 and its downstream target genes were highly expressed in most pancreatic cancer tissues（8/12）.Analysis of NRF2 expression and clinical data of patients showed that the expression level of NRF2 was related to the pathological grade and disease stage of patients with primary pancreatic cancer,but had no significant correlation with gender,age,lymph node invasion and distant organ metastasis.The expression level of total NRF2 in pancreatic tissues was significantly correlated with the CA19-9 level of patients.NRF2expression level was significantly correlated with overall survival and progression-free survival of patients.Univariate and multivariate analysis showed that NRF2 expression level was one of the risk factors affecting the prognosis of patients with pancreatic cancer.NRF2 expression level can better predict the prognosis of patients.2.Loss of Nrf2 promotes pancreatic tumorigenesis in miceThe survival time and body weight of KCN mice were significantly lower than those of KC mice（P<0.05）,but there was no significant difference between C and CN mice.There were no significant differences in fasted blood glucose and pancreatic weight among all groups.There was no significant difference in serum amylase in each group.HE and CK-19 staining showed that KC mice and KCN mice had obvious acinar ductal metaplasia pathological changes,and KCN mice had more and more serious acinar ductal metaplasia pathological changes than KC mice,while C mice and CN mice did not have obvious lesions at 4 weeks.At 8 weeks of age,KC mice had no obvious Pan IN lesions,and its ADM lesions was worse than before,but the obvious Pan IN lesions were found in the KCN mices,and pancreatic histopathology of N mice and CN mice showed no obvious Pan IN lesions.At 16 weeks of age,both KC and KCN mice showed obvious intraepithelial neoplasia.Compared with KC mice,KCN mice had a higher pathological grade and a wider range of Pan IN lesions,followed by KC mice.Some CN mice（1/5）also showed Pan IN lesions,while C mice had no obvious pancreatic lesions.Ki-67immunohistochemical staining showed that with the increase of weeks of age,KCN mice had obvious atypia of pancreatic ductal epithelial cells,and the proliferation ability of ductal epithelial cells was significantly enhanced,followed by KC mice.NQO-1immunohistochemical staining and q PCR results showed that:The m RNA and protein levels of Gclc,Gclm,Nqo-1 and Ho-1 in Nrf2 deficient mice（CN and KCN mice）were significantly lower than those in other two groups（C and KC mice）（P<0.05）.The decrease was more obvious in KCN mice（P<0.01）.At 8 weeks and 16 weeks of age,the m RNA expression levels of Il-1β,α-Sma and Il-6 in pancreatic tissues of KCN mice were significantly higher than those of other groups at the same week of age,suggesting that the activation of pancreatic stellate cells was obvious.8-OHd G immunohistochemical staining showed that KCN mice had more serious DNA oxidative damage than KC mice at the same week of age.The expression levels of EGFR,p-EGFR,PI3K,p-PI3K,AKT and p-Akt in pancreatic tissues of 16-week-old KC and KCN mice were significantly increased,especially in KCN mice,suggesting that the EGFR/PI3K/AKT pathway was highly activated in pancreatic tissues of KCN mice,thereby promoting tumorigenesis.3.NRF2 is highly expressed in pancreatic cancer cell lines,promoting the proliferation,migration and invasion of tumor cells,and playing a role in promoting the progression of pancreatic cancer.Cell experiments confirmed that NRF2 was significantly overexpressed in three human pancreatic cancer cell lines,in which CAPAN2 was the most significantly increased,followed by As PC-1,PANC-1.NRF2 and KEAP1 were stably silenced by lentivirus,and the results of cell counting,cell plate cloning and Ed U fluorescence staining showed that overexpression of NRF2 could significantly enhance cell proliferation,while inhibition of NRF2 could decrease cell proliferation（P<0.05）.WB results showed that NRF2 could promote tumor cell proliferation through IRS/PI3K/AKT/mTOR pathway.The results of second-generation sequencing showed the correlation between NRF2 and EMT-related genes and transcription factors.The results of cell scratch assay and Transwell chamber invasion assay showed that the migration and invasion ability of PDAC cells were significantly enhanced after overexpression of NRF2,and significantly inhibited after silencing NRF2（P<0.05）.q PCR and Western blot confirmed that overexpression of NRF2significantly promoted the expression of EMT-related proteins and transcription factors in PDAC cells.Second-generation sequencing results showed that:NRF2 is related to the expression of glycolysis-related genes.Subsequently,q PCR and Western blot experiments were used to confirm that inhibition of NRF2 could significantly reduce the expression of glycolysis-related genes and proteins in cells.Meanwhile,ECAR experiments were used to prove that NRF2 knockdown could significantly reduce the glycolysis rate.It suggested that NRF2 promoted the occurrence of tumor glycolysis.Then promote cell malignant biological behavior.Conclusion:1.NRF2 is highly expressed in pancreatic cancer tissues,and has significant correlated with serum CA19-9 level,histopathological grade and prognosis of patients,and can be used as a risk factor for poor prognosis of patients.2.Nrf2 inhibits the pancreatic tumorigenesis in mice.3.NRF2 is highly expressed in pancreatic cancer cell lines and promotes the proliferation of tumor cells through the IRS/PI3K/AKT/mTOR pathway,and promotes the migration,invasion and glycolysis of tumor cells,thus regulating the malignant biological behavior of pancreatic cancer.