Association Between PGC TagSNPs And Susceptibility Of Breast Cancer And Its Molecular Mechanism

Objective:Breast cancer is the most common cancer among women in China.The incidence of breast cancer is increasing,which seriously threatens women’s physical and mental health.In the last few years,research on genetic and genetic polymorphisms and the risk of breast cancer has been increasing.Studies have shown that Gene single nucleotide polymorphism(single nucleotide polymorphism,SNP)is one of the causes of individual differences in cancer.SNPs are the most common form of genetic mutation.As a genetic marker,SNPs can discover the role of genes in the occurrence and development of cancer and assess the risk factors of cancer.PGC is synthesized by the main cells of gastric mucosa and secreted into the gastric cavity.Under normal physiological conditions,PGC,as an end product of mature and differentiated gastric mucosal cells,is mainly expressed in the stomach.In recent years,more and more studies have shown that there is a close relationship between PGC and the occurrence and development of cancer.The research on ectopic PGC expression is increasing year by year.Especially in hormone-related tumors,such as prostate cancer,breast cancer and ovarian cancer,the expression of PGC in malignant tissues is significantly higher than that in normal and benign tissues.The PGC gene encoding PGC protein is located on chromosome 6,6p21.3-21.1.He et al.selected eight tagSNPs to effectively evaluate the occurrence and development of gastric cancer.The results indicate that the SNPs of PGC gene play a certain role in the occurrence and development of gastric diseases.At present,there is no related study about the role of PGC gene polymorphism in the occurrence and development of breast cancer.Therefore,this study intends to study the effect of PGC gene polymorphism on breast cancer risk in vivo by detecting seven tagSNPs of PGC gene,at the same time,immunohistochemistry and serum ELISA is used in breast benign tumors and malignant tumors.We studied whether the polymorphism of PGC gene could affect the expression of PGC protein and then be related to the risk of breast cancer.On the basis of defining the relationship between susceptibility gene polymorphism and risk of breast cancer,we further validated in vitro the relationship between PGC gene polymorphism and biological behavior of breast cancer cell lines and the molecular mechanism of regulating disease occurrence,so as to provide more evidence for the genetic mechanism of breast cancer at the gene level.Methods:According to the latest data published by NCBI dbSNP and HapMap databases,the labeled SNPs(tagSNPs)with the minimum allele frequency(MAF)>5%in Chinese population were selected to study by using software prediction method.Finally,five tagSNPs,PGC rs6912200,PGC rs6458238,PGC rs6941539,PGC rs9471643 and PGC rs3789210,were determined to explore their correlation with the risk of breast cancer and clinical biological behavior.Five polymorphisms of PGC gene rs6912200,PGC rs6458238,PGC rs6941539,PGC rs9471643 and PGC rs3789210 were detected by KASP in 544 cases of breast cancer and 536 cases of benign breast control group.Multivariate logistic regression was used to analyze the correlation between the polymorphism of PGC gene and the risk of breast cancer and its clinicopathological parameters.The online SHEsis method was used for haplotype analysis.Immunohistochemistry was used to detect the expression of PGC protein in breast cancer and benign breast group,and the difference of PGC protein expression between the two groups was compared.After that,the correlation between the expression of PGC and pathological parameters of breast cancer was analyzed.Finally,the difference of situ expression of PGC among different genotypes of tagSNPs of PGC gene was compared.The expression of serum PGC in breast cancer and benign breast control group was detected by enzyme-linked immunosorbent assay(ELISA),and the difference of serum PGC expression between the two groups was compared.Then the correlation between serum PGC protein expression and pathological parameters of breast cancer was analyzed.Finally,the difference of serum PGC protein expression between different genotypes of PGCtagSNPs was compared.Luciferase activity of reporter plasmids of different genotypes at rs9471643locus of PGC gene was detected by luciferase reporter gene system,and the promoter activity of different alleles was compared.Luciferase activity of reporter plasmids of different genotypes at rs9471643locus of PGC gene was detected by luciferase reporter gene system,and the promoter activity of different alleles was compared.Results:1、The analysis of the correlation between tagSNPs typing of five PGC genes and the risk of breast cancer showed that homozygous mutation and recessive model of rs9471643 locus of PGC gene increased the risk of breast cancer in the general population(P<0.05).Heterozygous carriers of rs3789210 locus could increase the risk of breast cancer(P=0.045).In younger group,rs6912200 CC/TC+TT can increased the risk of breast cancer.And in thinner group,rs9471643 CC type can increase the risk of BC.In fatter group,rs3789210 GC/GG+GC can increase BC.2、The relationship between PGC tagSNPs and the clinicopathological parameters of breast cancer showed that the maximal diameter(>2cm)of PGC gene was lower than that of breast cancer(P=0.047).The malignancy of breast cancer with positive HER2expression was relatively low in the carriers of mutation T allele of rs6912200(P=0.048).There was a positive correlation between ER negative and malignancy of breast cancer(all P values<0.05).The recessive model of rs3789210 locus showed a positive correlation between PR negative and malignant degree of breast cancer(P=0.045).In carriers of G allele of rs3789210 locus,HER2 positivity was positively correlated with breast cancer malignancy(all P values<0.05).3、In older group,tumor size was correlated with BC in rs6912200 C allele carrier.In homozygote TT gene carriers at rs6941539 locus,ER negative was positively correlated with breast cancer malignancy(all P values<0.05);in homozygote TT gene carriers,PR negative was positively correlated with breast cancer malignancy(all P values<0.05);As for rs3789210 GG,the deficiency of expression of PR was positively correlated with BC,and to G allele carrier,HER2 positive and high-expression of Ki-67 was also positively correlated with breast cancer malignancy.rs6941539 T allele carriers were significantly correlated with histological types of breast cancer.In the group of fatter ones,rs6912200 C allele carriers were correlated with metastasis of lymph and HER2status.And rs9471643 C allele carriers was related to metastasis of lymph.As for rs3789210 GG carriers,negative expression of ER and PR were positively correlated with BC,and G allele carriers with high-expression of Ki-67 were also positively correlated with BC.4、Haplotype analysis of the 5 polymorphic loci found that carrying C-G-C-C-G(rs6912200-rs6458238-rs6941539-rs9471643-rs3789210)polymorphism significantly increased the risk of breast cancer(P=0.002).5、In situ protein expression,we found that the immunohistochemical score of PGC protein expression in the breast cancer group was significantly higher than that in benign grou.Further analysis of the relationship between PGC protein expression and the clinicopathological parameters of breast cancer showed that the PGC protein expression was correlated with the HER2 state in the larger BMI group.And in the lower BMI group,the PGC protein expression was correlated with TNM stage.6、As for breast cancer patients with positive ER expression and PR expression,serum expression of PGC was significantly higher than that of the patients with negative ER and PR expression(all P values<0.05).At the level of serum protein expression,we found that the expression of serum PGC in the breast cancer group was significantly higher than that in the benign breast group.Further analyzes show that in the general population,serum expression of high histological grade(level III)was lower than lower histological grade(less than level II)in breast cancer(P=0.008).The positive expression of ER and PR expression positive breast cancer patients,the serum PGC expression is significantly higher than the two negative expression(all P values<0.05)。Furthermore,in younger group and thinner group,the serum PGC expression trend was consistent with the general population trend in histological grading,expression of ER and PR(all P values<0.05)。In younger group,serum expression of PGC in high histological grade was significantly lower than that in lower grade breast cancer.In fatter group,expression of PGC in patients with PR deficiency was significantly lower than that in patients with PR positive.7、In breast cancer,the expression level of GG-type at rs9471643 locus was significantly higher than of GC type or GC+CC type(all P<0.05).In benign group,the expression level of CC type at rs3789210 locus was significantly higher than that of GG or CC+GC type(all P<0.001).8.In the overall analysis,we found that the serum expression level of GG type at rs6458238 of PGC gene was significantly higher than that of GA type or GA+AA type(all P values<0.05).Further analysis showed that in the benign breast group,the serum expression level of rs6458238 loci was consistent with the trend in the overall analysis,which means the serum expression level of GG type protein was significantly higher than that of GA type or GA+AA type(all P values<0.05).9、In vitro experiments,luciferase reporter results of promoter showed that luciferase activity of mutant C allele was significantly reduced compared with rs9471643 polymorphic wild type G allele,(P=0.0089).Conclusion:1、The rs9471643 and rs3789210 loci of PGC gene may increase the risk of breast cancer in the whole population.The rs6912200 loci increased the risk of breast cancer among people younger than 40 years old.When BMI was less than 24kg m~2,rs9471643 loci can increases the risk of breast cancer.When BMI was greater than 24kg/m~2,rs9471643 loci can increases the risk of breast cancer.2、There were correlations between PGC gene rs6912200,rs6941539 and rs3789210 loci and breast cancer pathological parameters.The rs6912200,rs6458238,rs6941539 and rs3789210loci were correlated with pathological parameters of breast cancer in people older than40 years old,and rs6941539 loci were correlated with pathological parameters of breast cancer in people younger than 40 years old.The rs6912200,rs9471643 and rs3789210 were correlated with pathological parameters of breast cancer in people with greater BMI.The rs6458238 and rs6941539 were correlated with pathological parameters of breast cancer in people with less BMI.3、Haplotype analysis of the 5polymorphic loci C-G-C-C-G(rs6912200-rs6458238-rs6941539-rs9471643-rs3789210)significantly increased the risk of breast cancer.4、The expression of PGC in situ in breast cancer was higher than that in benign breast control group.At the same time,the expression of PGC protein in situ was correlated with HER2 status and TNM staging in breast cancer;5、The expression of serum PGC in breast cancer group was higher than that in benign breast control group.Meanwhile,serum PGC expression was correlated with histological grade,ER expression,PR expression and TNM stage of breast cancer.;6、The rs9471643 locus in the promoter region of PGC gene can decrease the promoter activity and down-regulate the expression of PGC protein in situ,further clarifying the possible mechanism between rs9471643 locus and the risk of breast cancer.7、The carrier of GG genotype at rs3789210 locus in benign breast group was associated with the decrease of in situ expression of PGC protein,and the carrier of A allele at rs6458238 locus was associated with the decrease of in situ expression of PGC protein in general population and benign breast group.