A NMBA-induced Esophageal Precancerous Lesion Mechanism Of DNA Polymerase Beta Knockout Mice

Background and Purpose Esophageal cancer(EC)is a common malignant tumor of digestive tract.It mainly includes two kinds of pathological types,esophageal squamous cell carcinoma(ESCC)which originates from esophageal epithelial cells,and esophageal adenocarcinoma(EAC)originating from esophageal submucosal gland or gastric cardia gland.Notably,almost half of the global EC occur in China,and most patients are the first pathological type.The etiology of EC is complex,with high mortality,poor recovery after treatment and poor quality of life.Early screening of EC has a good prognosis.Therefore,early prevention of esophageal precancerous lesions by chemoprevention is an effective way to reduce the mortality and improve the prognosis of EC.The previous study suggest that there is a high expression and mutation of DNA Polymerase ?(Polb)in EC.The abnormality of Polb may play an important role in the development of EC.Therefore,we will further explore the relationship between Polb and EC,and hope to find a new way for chemoprevention of EC.Polb(11)a DNA repair enzyme,plays a key role in Base-Excision Repair(BER).Polb exists in eukaryotes and it is a conserved housekeeping gene with DNA polymerase and lyase.It can repair damaged DNA.Lack of 3'-5'exonuclease activity,Polb cannot timely correct the mismatch bases,which increased DNA mismatch rate.Therefore,Polb is the lowest fidelity DNA polymerase.In addition,Polb is widely involved in the process of DNA damage synthesis,DNA recombination and chemotherapeutic drug resistance.The biological function of Polb determines its importance and necessity.The whole body knockout of Polb leads to death in mice.On the one hand,this phenomenon indicates that Polb has a great influence on embryonic development.On the other hand,it suggests that it is urgent for researchers to explore new animal models for clarifying the function of Polb in cell.This study is intended to construct an animal model of Polb deficiency.Among many methods of gene knock-out animal model,the Cre-Loxp Polymerase recombination system was used here.This is a conditional gene targeting technique that can target the Polb gene in the epithelial cell of the esophagus.It can avoid the lethality of the whole body knockout,but also ingeniously reduces the effect of Polb gene deletion in other tissues.Furthermore,on the basis of the Polb knockout model,esophageal precancerous lesions was induced by N-nitrosomethylbenzylamine(NMBA)to further study the relationship between Polb and the development of esophageal cancer.Finally,we use m RNA expression microarray to analyze the mechanism of esophageal carcinogenesis in Polb knockout mice.We will preliminarily discuss which genes will change after Polb gene deletion,and find out molecules related to Polb and esophageal carcinogenesis.In summary,knockdown of Polb gene in mouse esophageal epithelium by Cre-Loxp recombination system firstly.Then NMBA was used to induce Polbknockout mice to develop esophageal precancerous lesions,and a reliable tissue-specific Polb knock-out animal model of esophageal precancerous lesions has been established.The purpose of this study is to explore the role of Polb in the pathogenesis of esophageal precancerous lesions and provide a new target for chemoprevention of esophageal precancerous lesion.Materials and methods1.The Polb specific knockout mouse model of esophageal tissue was established by Cre-Loxp system.PolbLoxp Loxp mice(Polb mice)and Cre transgenic mice(ED-L2-Cre mice)were paired to produce offspring.The genotype of mice were identified by extracting DNA from mouse tails.The genotype of ED-L2-Cre;PolbLoxp/Loxp was the knockout mice(Polb-KO mice).2.Comparison with Polb-Loxp mice(n=8)in the same nest,Polb-KO mice(n=8)were selected.1 week after birth,the weight of mice began to be weighed.Acquisition of esophageal tissue in 4 weeks old mice.Paraffin embedded,sectioning,and hematoxylin eosin(H&E)staining.Morphological changes of esophageal epithelial cells were observed by optical microscope.3.The RNA of mouse esophageal epithelial tissue was extracted.After reversing to c DNA,Polb gene was amplified by Q-PCR.The transcription level of Polb in mouse esophageal epithelium was detected by this method.Extraction of protein from mouse esophageal epithelial tissue.The protein expression level of Polb in mouse esophageal epithelium was detected by Western blotting assay.Immunohistochemistry was used to detect the expression level of Polb in the esophageal epithelium of knockout mice,and the effect of knockout was verified at various levels.4.Objective to establish a NMBA induced Polb knockout model of esophageal cancer in mice.Group of experimental animals: Polb-KO mice and Polb-Loxp mice(7~9 weeks old)were divided into two groups according to genotype.(1)MBA+Polb-Loxp mice(n=15): the genotype of mice was Cre-/-;PolbLoxp/Loxp and was subcutaneously injected with NMBA(1 mg/kg),3 times a week for 5weeks.(2)NMBA+Polb-KO mice(n=15): the genotype of mice was ED-L2-Cre;PolbLoxp/Loxp and was subcutaneously injected with NMBA(1 mg/kg),3 times a week for 5 weeks.The weight of mice was tested every week.At 20 th and 40 th weeks,esophageal lesions were observed by random sampling.The whole experiment continued to be observed until the end of the 51 th week.Esophagus of mice were taken after the experiment.A part of the mouse esophagus was used for HE staining in paraffin blocks.The histological changes of esophagus and pathological classification of esophageal lesions were observed under optical microscope.Another part of mice esophagus was separated from esophageal epithelium mucosa,and RNA was extracted to make m RNA expression microarray.5.The results of m RNA expression spectrum chip were analyzed.The difference genes between the two groups were selected.Then preliminary explored the types of cell biological functions involved in differential genes.6.In order to preliminarily verify the results of the chip,immunohistochemistry was used to detect protein expression levels of Ki67,PI3 K,p-AKT,p-ERK and PLK1 in two groups of mice.Results1.PCR results showed that both 400 bp PolbLoxp bands and 199 bp Cre bands were detected in the mice,which were called Polb-KO mice.There was no significant difference in body weight of Polb-KO mice from 1 week to 4 weeks after birth,compared with Polb-Loxp mice(P>0.05).HE staining of mouse esophagus was observed by optical microscope.Compared with Polb-Loxp mice,the morphology and structure of esophageal epithelial cells in Polb-KO mice did not change significantly.2.Q-PCR results showed the m RNA transcription level of Polb gene in Polb-KO mice was significantly reduced,compared with the Polb-Loxp mice(P<0.05).Western blot results showed that the expression level of Polb in esophageal epithelial tissue of Polb-KO mice was significantly lower than Polb-Loxp mice(P<0.05).Immunohistochemical staining showed that the expression of Polb in esophageal epithelial cells of Polb-KO mice was significantly lower than Polb-Loxp mice.3.In constructing NMBA-induced Polb-KO mouse model of esophageal precancerous lesions,there was no difference in body weight between the two groups(P>0.05).Compared with the NMBA+Polb-Loxp group,the number of lesions in the esophageal epithelial tissue of NMBA+Polb-KO group was larger (P<0.05).After pathological grading of the esophageal lesions in mice,the statistical results showed that severe atypical hyperplasia was found only in the NMBA+Polb-KO group.It suggests that Polb-KO mice are more sensitive to NMBA-induced esophageal carcinogenesis.4.The results of the m RNA transcriptome chip show that there were 504 differentially expressed genes among the two groups,including 208 up-regulated genes and 296 down-regulated genes.These differential genes involved cell proliferation,cell cycle and so on.Among them,proteins related to cell proliferation involve PI3K-AKT,MAPK-ERK,PLK1/CENP-A and other signal pathways.5.Immunohistochemistry results showed the positive cell rates of Ki67,PI3 K,p-AKT,p-ERK and PLK1 in the esophageal epithelial cells in NMBA+Polb-KO knockout mice increased significantly,compared with the NMBA+Polb-Loxp group.Conclusion The animal models of esophageal precancerous lesions induced by NMBA were successfully established in Polb knockout mice.The process of esophageal carcinogenesis,namely,atypical hyperplasia of esophageal epithelial cells,may occur by activating PI3K-AKT,MAPK-ERK,PLK1/CENP-A pathways.