The Promoter Methylation Of SIRT6 And FOXO3A Genes In Residents From Bama County Of Guangxi

Objective: To investigate the methylation levels of Cp G island of SIRT6, FOXO3 A genes promoter in residents lived in Bama county of Guangxi. To discuss the relationship between methylation levels of these two genes with the phenomenon of longevity in Bama combined with the early results of m RNA and protein expression levels, and to provide theoretical basis for further exploration of Bama longevity phenomenon.Methods: Samples were collected in May of 2011 in Bama Yao nationality autonomous county of Guangxi, whom were defined as long-lived group(LL group) and non-long-lived group(NLL group) according to the definition of longevity family history. 129 healthy residents with longevity family history composed the longevity-lived group, then further divided into longevity group and offspring group. Longevity group include 42 subjects and offspring group include 87 subjects. 86 residents without family history of longevity, composed non-long-lived group. Blood samples were collected from the study objects, and genomic DNA were extracted. Pyrosequencing method was applied to detect methylation levels of SIRT6 and FOXO3 A gene promoter region. Combined with the early result of gene m RNA and protein expression. Chi-square test was applied to analysis the basic information of the study objects and rank and inspection to compare the gene methylation rate of groups. T or chi-square test to compare the m RNA and protein level. Logistic regression to analysis the relationship of methylation level and longevity. Spearman correlation analysis to the relationship between methylation rate and age, m RNA and protein expression. Multiple linear regression to analysis the relationship of gene methylation level and smoking, dringking.Results: 1. The basic information of study objects: The total study population was 215, of long-lived group 129 and non-long-lived group 86. There was no significant difference of the sex and there was significant difference of the age between LL group and NLL group. The smoking rates were 24.0% and 27.9% and the drinking rates were 69.0% and 65.1% respectively in LL group and NLL group, and there weren’t significant differences of the smoking rates and the drinking rates(P > 0.05).2. The comparison of methylation levels, m RNA and protein expression: The methylation level of SIRT6 was lower in LL group when compared with NLL group. And the methylation level of p2 and p5 in SIRT6 were significantly different between LL group and NLL group(P<0.05). The LL group was lower than NLL group. The m RNA level was higher(P<0.05)and the protein expression was not statistical different(P>0.05)when LL group compared with NLL group. There were significant difference of p2 and p5 of SIRT6 after the LL group was divided into longevity group and offspring group(P<0.05). The methylation rate of p2 was lower when the longevity group and offspring group compared with NLL group(P<0.05) and the methylation rate of p5 was lower when offspring group compared with NLL group(P<0.05), while the other locus of SIRT6 weren’t significantly different between the three groups. The m RNA level of SIRT6 in offspring group was higher than longevity group and NLL group. The protein expression of SIRT6 was highest in the longevity group and was lowest in the offspring group.The methylation level of p3 in LL group was higher than NLL group and the methylation level of p5 of FOXO3 A was lower than NLL group(P<0.05).The m RNA level was higher(P<0.05) and the protein expression was not statistical different(P>0.05) when LL group compared with NLL group. The methylation levels of p3, p4 and p5 locus of FOXO3 A gene were obviously different between the three groups after the LL group was divided into longevity group and offspring group(P<0.05).The methylation levels of p3 and p4 were lower and the methylation rate of p5 of FOXO3 A was higher when offspring group compared with NLL group(P<0.05).The m RNA level of FOXO3 A was higher in offspring group compared with the longevity group and NLL group. The protein expression of FOXO3 A was highest in the longevity group and was lowest in the offspring group.3. Logistic regression analysis of all the methylation locis: Logistic regression analysis result showed than the p2 and p5 locus of SIRT6 were the protective factors of longevity. The OR value and 95% CI of the p2 and p5 were respectively 0.411(0.218~0.218) and 0.218(0.708~0.708) after the adjustment of gender, age, smoking and dringking. The p3 loci of FOXO3 A was the protective factors of longevity and the OR value and 95% CI were 0.808(0.699~0.934),others showed no relationship with longevity.4. The correlation of gene methylation levels and age, m RNA expression and protein expression: Of SIRT6 gene, the second Cp G loci p2 was negatively correlated with age(P<0.05). P2, p5 and pm were negatively correlated with m RNA expression while p4, p5 and pm of were positively correlated with protein expression(P<0.05). P5 of FOXO3 A was positively correlated with age(P<0.05). P1, p2, p4 and pm were negative correlation with m RNA expression while p4 and pm were positive correlation with protein expression(P < 0.05).5. The p3 loci of SIRT6 gene was related to drinking(P < 0.05) and the other locus weren’t related to smoking and drinking(P > 0.05). All the five locus of FOXO3 A gene were no relations with smoking and dringking(P > 0.05).Conclusion: 1. Part sites of SIRT6, FOXO3 A gene methylation levels may be associated with the longevity phenomenon of Guangxi Bama region.Their low levels of methylation may be the favorable factors that affect longevity.2. The relationship of SIRT6, FOXO3 A gene methylation level and age was different depending on different sites. The methylation level of part sites were negatively related to the m RNA expression and part were positively related to protein expression.3. The methylation levels of SIRT6 and FOXO3 A gene were little related with smoking and drinking alcohol.