The Function And Mechanism Of Pepsinogen C Gene Insertion/deletion Polymorphism In Regulating Gene Expression

Objective: Human pepsinogen C(PGC)is a precursor of pepsin which is stomach specific functional enzyme.PGC is an endo protease which belongs to aspartic protease family and is a final product of gastric mucosa cell differentiation and maturation.There is a 100 bp insert/deletion polymorphic fragment located between area of PGC gene exon 7 and 8.Patients with a homozygous PGC allele1 genotype have an increased risk of atrophic gastritis and gastric cancer(GC).PGC gene insert/deletion polymorphism had significant association with genetic susceptibility to gastric cancer,thus it can be used as a warning sign of individuals with high risk of GC.At present,the exact mechanism of PGC gene insert/deletion polymorphism impacting the susceptibility to gastric cancer is unclear and remains to be discussed.Some introns have the function to regulate the promoter activity of gene,others possess the promoter activity.According to DNA sequence analysis,PGC insertion/deletion polymorphism fragment contains the TATA box sequence,which plays a vital role in deciding the position of transcription initiation.A simple promoter could be formed by TATA box and transcription initiation position.Some specificity sequence containing the TATA box structure in introns region also has the function of promoter.The mutation of or changing the distance from the transcription start site could result in transcription starting in other position,thus affecting the gene expression in transcriptional level.So,do the insertions/deletions fragments located in PGC gene intron area with the TATA box sequence have promoter activity? In addition,TATA box of DNA sequence is more often the site transcription factor binds,which could participate in the interaction with transcription factor and modulate the transcriptional process.Whether PGC gene insertion/deletion polymorphism fragments have different promoter activity by influencing the transcription factor combination and lead to different promoter activity still need to be clarified.This study aims to clarify the function of PGC gene insertion/deletion polymorphism fragments in gene expression regulation through studying the promoter activity and transcription regulation function.We hope to systematically clarify the relationship PGC gene polymorphism and genetic susceptibility to GC as well as establish effective warning marker for individuals with high risk of GC and provide valuable experimental basis and theoretical reference.At the same time,in order to further explore the significance of combined detection of PGC and other gastric cancer markers,we investigate the value of combined expression of gastric mucosal differentiation protein PGC and gastric cancer(GC)associated antigen MG7 for diagnosis of GC and prediction of the development of GC from precancerous conditions.Method:1.The whole mRNA and 8-9 exons mRNA of different genotype of PGC gene were specifically amplified by real-time PCR and the effect of different length of PGC gene insertion/deletion polymorphism fragments on PGC gene expression was evaluated by immunohistochemistry method with different gastric mucosa tissue.2.We constructed insertion/deletion genotypes of PGC 4200 bp promoter region luciferase expression vector.Dual luciferase reporter gene systems were used to measure the promoter activity,comparative analysis of different promoter activity in different PGC gene insertion/deletion polymorphism segments.3.Transcription factors combined with PGC gene insertion/deletion polymorphism fragments were predicted using Bioinformatics information.Whether the PGC gene insertion/deletion polymorphism fragments could specifically bind to transcription factor was detected by EMSA.4.Levels of PGC and MG7 in biopsies were determined by SP immunohistochemical staining.ROC curve was drawn to calculate the diagnostic efficacy of gastric cancer and its precancerous lesions.Gastric mucosal biopsies were obtained and histopathologically examined for 285 subjects enrolled from a region with high incidence of GC.Subjects testing negative for GC(n=208)were monitored from 1998 to 2015.Results: 1.The expression of PGC m RNA level is significantly higher in non-gastric cancer group than that in the cancer group(F = 40.772).Patients with a homozygous PGC allele1 genotype had a significantly lower m RNA expression than that in heterozygous allele type 1(F = 2.038)and the other type of groups(F = 5.550);Individuals carried heterozygous allele 1 geno-type had a significantly lower m RNA than that in other groups(F = 3.503).In gastric cancer group,m RNA expression in homozygous PGC geno-type 1 was significantly lower than heterozygous allele type 1(F = 9.420)and other type of groups(F = 2.138);8-9 exons PGC gene expression rate has no statistically significant difference between the groups(F = 1.329,F = 1.866).From alleles 1 heterozygous type to other type to homozygous type,PGC protein positive expression rate was gradually decreased.Homozygous allele type 1 was statistically significant difference between the group and other groups(p = 0.009),whose OR was 2.114(95% CI 1.177 3.817)after adjustment for age and sex.Protein positive expression rate was gradually decreased in GS group;the allele 1 homozygous group was significantly lower than other groups.2.In different cell lines of AGS and SGC-7901,luciferase activity of PGC genes polymorphism short(436 bp)and long(310 bp)fragments have significant difference,and the ratio of long fragments are more than 3 times that in short fragments(p<0.05).However,luciferase activities of both groups were lower than negative controls.Either polymorphisms could enhance the 4200 bp luciferase activity(p<0.05).The ratio of insertion genotype with PGC gene promoter was higher than that of deletion genotype with PGC gene promoter(0.55:0.48).3.In the 7 cell lines(.BGC-823,NCI-N87,AGS,SGC-7901,MKN-45,GES-1,BGC-803),specific binding band with OCT-1 were found in GES-1,NCI-N87 and MKN-45,of which strong band was found for GES-1 and moderate and slight bands were found for NCI-N87 and MKN-45.It is indicated that the fragments which binds to OCT-1 probe was insertion PGC polymorphism fragment and insertion PGC polymorphism fragment have binding site for OCT-1.4.The positive expression rate of PGC gradually decreased from non-atrophic gastritis(GS,92.1%)?atrophic gastritis(GA,26.1%)? GC(0.0%).Positive expression rate of MG7 gradually increased from GS(15.0%)?GA(82.4%)?GC(94.8%).The differences among the groups were statistically significant(p<0.05).The “PGC+MG7-” was the dominant type in GS group,while “PGC-MG7+” was the dominant type in GA and,especially in GC groups.Compared with “PGC+MG7-”,“PGC-MG7+” showed 113.4-fold increased risk of GC(95%CI: 15.3-869.4,p<0.001).In the follow-up cohort,the risk of gastric cancer in the PGC-MG7+ group was higher than that in the other groups.Conclusion:PGC gene insertion/deletion polymorphism fragments could influence the expression of PGC gene in transcription and protein levels.PGC gene deletion polymorphism was inversely associated PGC expression.2.PGC gene insertion/deletion polymorphism fragments could modulate gene promoter activity.Individuals carried long PGC gene insertion polymorphism fragment may have stronger promoter activity than that carried the short fragment.3.PGC gene insertion polymorphism fragment could bind to transcription factor OCT-1 and may affect the promoter activity and thereby affect the expression of PGC.4.The “PGC–MG7+” can be employed as a useful follow-up panel for detecting high risk individuals of GC and their dynamic assessment that require multi-center large-scale validation in the further.