Impacts Of Genetic Variants In The Promoter Of HBB Gene On Its Serum MRNA Expression And Its Association With Metabolic Parameters

Background and aims: ?-globin(HBB)is an important component of hemoglobin.Previous studies mainly focus on the link between its abnormal expression and some single-gene disorders such as sickle cell anemia.With the advance of nuclear acid sequencing technology,HBB was found to express in other tissues besides red blood cells and maintain certain level in serum which has been linked to some age-related diseases such as atherosclerosis.However,the function and the biological significance of free serum HBB mRNA remain unexplained.Healthy long-lived cohort who have escaped most age-related disorders is regarded as an ideal population model for human aging study.This study investigated the relationship between HBB promoter polymorphism and serum HBB mRNA expression level,blood glucose and blood lipid and other metabolic indicators in long-lived population,so as to better understand the effect of HBB on human aging.Methods: Target proteins of HBB gene were predicted by String and pathway enrichment and function interpretation were analyzed by KEGG and GO.107 long-lived individuals(age ? 85 years),74 elderly(age 65-84 years)and 131 youngers(age ? 65 years)were recruited from Guangxi Hongshuihe River Basin in this study.All participants were tested for metabolic variables such as fasting blood glucose(FBG),lipids and creatinine(Cr);serum HBB mRNA was detected by RT-q PCR;the promoter of HBB gene was sequenced by PCRSanger to discover polymorphisms.Results: HBB gene targets 10 binding proteins,whose biological processes mainly focus on oxygen transportation,hydrogen peroxide catabolism and nitric oxide biosynthesis.Serum HBB mRNA level of long-lived group is significantly lower than that of non-long-lived group(P?0.05);Serum HBB mRNA level is associated with Apo A,HDL,FBG,Cr and Ig G;The6 SNPs are detected in the promoter of HBB gene,in which,the genotypic and allelic frequencies of rs74234654,rs35755129 are statistically different between long-lived and non-long-lived group,and the FBG and LDL levels of rs35755129 G allele in females of younger group were lower than that of non-G(AA genotype)carriers(P?0.05);the LDL and Ig G levels in males rs35755129 G carriers in longevity group and the FBG,Apo A,BUN and Cr in males of younger group are remarkably lower than that of G noncarriers(AA genotype)(P?0.05);The mRNA expression level of serum HBB in rs200771769 G/G genotype carriers in the longevity group was lower than that in A/G genotype carriers(P?0.05).Conclusions: Serum HBB mRNA level may be influenced by the polymorphisms in the promoter of HBB gene,the interaction between HBB mRNA and metabolic risks may affect the pathogenesis of age-related diseases and aging process.Chapter 1 Exploration for the potential functions of HBBBackground As a critical component of hemoglobin,HBB is more important type of globin in adults.Although it is known that HBB mainly play a function role at the hemoglobin level,recent studies have found that the mRNA of HBB can be stable in the serum,and the expression in the serum of the elderly black women and the youth is different,and the relevant mechanism of action is still unknown.In tumor studies,it has been found that HBB has certain antioxidant effect,which can reduce the damage of ROS to cells.According to the aging free radical theory,ROS production is an important factor to accelerate the aging of the body.Therefore,the difference in the expression of HBB mRNA in different age groups is most likely related to the aging state of the body.The emergence of medical biological information technology can not only be used as a tool to analyze experimental results,but also provide theoretical support for experimental exploration.With the help of databases and analytical tools such as STRING KEGG GO,co-expressed genes are mined and relevant functional annotations are made to explain the role of certain genes in a biological process.Objectives The potential functional characteristics of HBB were mined by bioinformatics,so as to provide theoretical support for the subsequent experimental studies on the relationship between HBB and aging.Methods 1.STRING conducts the prediction of HBB targeted proteins,finds out the interacting proteins,and further understands the possible functions of HBB by understanding the function of the interacting proteins.2.KEGG enriched the pathway in which HBB was involved,and found the proteins co-expressed with HBB in the enrichment pathway.Biological processes,cell components and molecular functions of HBB were annotated by GO,so as to understand the main biological processes and existing sites involved in HBB.Results 1.STRING predicted that there are ten HBB target binding proteins,in addition to a variety of hemoglobin,it also includes Apo A1,CAMP and other immune metabolites.2.KEGG results: the HBB enrichment pathways were African trypanosomiasis and Malaria,and the proteins that interact with HBB in the above pathways include TNF,TGFB1 inflammatory proteins in addition to various hemoglobin proteins and Apo A1.3.GO analysis results: HBB is involved in the biological processes related to oxidative stress,such as oxygen transport,hydrogen peroxide decomposition and positive regulation of nitric oxide.The molecular functions of target genes are mainly the synthesis and transportation of substances.Target genes are distributed in the blood system as well as inside and outside the cell.Conclusions HBB is found to distribute in the blood system and other body cells.HBB not only combines with hemoglobin,but also interacts with a variety of immunoglobulins and inflammatory cytokines.Furthermore,HBB regulates several oxidation-related biological processes such as oxygen transportation,the degradation of H2O2 and the synthesis of NO,which is the basis of following studies.Chapter 2 The expression of HBB mRNA in long-lived cohort and its impacts on metabolic profilesBackground Aging is universal process in which body integrity gradually loses and biological functions degenerate.Aging is also a complicated phenotype which is determined by both genetic and environmental factors.During aging,with the disruption of body homeostasis,human beings are prone to age-related disorders like cardio-cerebral,autoimmune,Alzheimer's diseases and cancers.Unfortunately,the mechanisms underlying these age-related disorders and aging process remain poorly understood.Very recently,some free RNAs were noted to present in human serum,in which HBB mRNA,transcribed from ?-globin gene,was found to express significantly higher in the elderly as compared to younger counterparts.However,its biological significance and potential impacts on aging process remain largely unknown.Objectives To determine the serum HBB mRNA expression in long-lived,i.e.,healthy aging individuals and analyze its relationship with metabolic risks,in an attempt to further understand the biological significance of HBB mRNA in human aging.Methods One hundred and seven long-lived individuals(age ? 85 years),74 elderly(65 ? age ? 85 years)and 131 youngers(age ? 65 years)were recruited from Guangxi Hongshuihe River Basin in this study.Metabolic parameters like fasting blood glucose(FBG),lipid panel and homocysteine(Hcy)were tested by routine commercial kits and serum HBB mRNA level was measured by RT-q PCR its association with metabolic profiles was analyzed.Results 1.Serum HBB mRNA level of long-lived group is significantly lower than that of elderly and younger group(P?0.05);2.Serum HBB mRNA level is positively linked to Apo A,HDL and FBG while negatively associated with Cr,Hcy and Ig G.Conclusions The under-expression of serum HBB mRNA and its unique associations with metabolic risks in long-lived individuals from Hongshuihe River Basin may partially interpret their lower morbidity of age-related diseases and longer lifespan.Chapter 3 Effects of HBB promoter polymorphism on serum mRNA and metabolic parametersBackground HBB is one of the important globin in adults,whose functional or expressional abnormality leads to inherited blood disease ?-thalassemia.The expression of mRNA is regulated by elements such as promoter and enhancer and the regulation of promoter seems more important.Objectives To explore the genetic variants in the promoter of HBB gene and their associations with metabolic parameters.Methods DNA was extracted from peripheral white blood cells and the promoter of HBB gene was sequenced by PCR-Sanger to discover possible polymorphic loci.Results 1.Six SNPs,rs10742583,rs10742584,rs200771769,rs74234654,rs10768684 and rs35755129 were detected in the promoter of HBB gene,all of which are compatible with Hardy-Weinberg equilibrium;2.The genotypic and allelic frequencies of rs74234654 and rs35755129 were statistically different between long-lived and non-long-lived groups(P ? 0.05);3.The serum HBB mRNA level of rs200771769 G/G genotype carriers in long-lived group was significantly lower than that of A/G carriers(P ? 0.05);4.The FBG and LDL levels of rs35755129 AG/GG carriers in females of younger group were lower than that of AA carriers;LDL and Ig G levels in males harboring AG/GG genotype in long-lived group and FBG,Apo A,BUN and Ig G levels of AG/GG carriers in younger group are marked lower than that of AA carriers(P ? 0.05 for all).Conclusions Polymorphisms in the promoter of HBB gene affect the serum expression of HBB mRNA and link to several metabolic variables in a gender specific manner.