Menopausal Age-related Depression,Gene Methylation,and Preliminary Mechanism

Objective: To screen and validate the methylated status of menopausal age-related genes,and explore the association between the methylated status of those and menopausal age-related depression.whether the methylated genes are one of important underling mechanisms in the expression changes of corresponding proteins.Methods: 1)The menopausal age of the study subjects was ≤48 years old for the early menopause group,49 to 51 years old for the control group,and ≥52 years for the late menopause group.The differential methylated gene between early and late menopausal women was screened by Illumina Human Methylation 450 K chip from genomic DNA of adipose tissue.According to this study’s destination,we select some candidate genes for further investigation.We use GO database to analyze the functions,biological processes and cellular components of the candidate genes;use KEGG for signal pathway analysis;and reveal the gene-encoded proteins potential interaction by STRING Interaction Network.2)Between January 2015 and December 2018,251 postmenopausal women were enrolled in the gynaecological hospitalization of Hangzhou Hospital affiliated to Nanjing Medical University,including the early menopause group（n =80）,the late menopause group（n =82）,and the control group（n =89）.Three milliliters of peripheral venous blood was drawn,and EDTA·K₂ was used to anti-coagulate.In addition,3 pieces of soybean-sized subcutaneous adipose tissue were collected at the surgical incision from some patients during the surgical treatment（early menopause group / n = 28,late menopausal group / n = 25,and control group / n = 25）.All subjects were evaluated by DSM-IV depression diagnostic criteria and the Hamilton Depression Assessment Scale（HAMD-17）was used to assess the severity of depression.The genomic DNA was treated by the Epi Tect Bisulfite Kit.Three candidate genes including BDNF,CACNA1C,and ARHGEF10 were detected in adipose tissue and peripheral blood by methylation-specific quantitative PCR（MethyLight）.The results of correlation in two samples were tested.Meanwhile,we assessed the association between the genes methylation status in peripheral blood and the severity of depression.Additionally,real time PCR and western blotting were used to detect the expression of mRNAs and proteins in adipose tissue,respectively.Results: 1)The results of methylation chip showed that 415 differential methylation gene loci were obtained in early menopausal women,compared with late menopausal women,of which 169 were hypermethylated gene loci and 246 were hypomethylated gene loci.Combining of the literature and subsequent studies’ needing,we selected 3genes with significantly hypermethylation（BDNF,ARHGEF10,and CACNA1C）as candidates for this study.Through bioinformatics analysis,they may be involved in biological processes such as nerve cell growth and providing nutrition,hormone or neurotransmitter release,regulation of cell proliferation and apoptosis.2)The status of BDNF and CACNA1C in adipose tissue and whole blood were obviously hypermethylated in the early menopause group comparing with the control group and late menopause group,while the methylated status of ARHGEF10 was not significant among three groups.The PMR（percentage of methylation ratio）of BDNF,CACNA1C and ARHGEF10 in adipose tissue and blood exhibited a good correlation（γ=0.625,P <0.001;γ=0.553,P =0.003;γ=0.571,P <0.001）.The high-PMR of BDNF methylation in blood was significantly associated with moderate or severe depression in early menopause group（x² =4.236,P =0.039）,and the risk of high-PMR carrier was 1.912 times than that of low-PMR carrier.The PMRs of ARHGEF10 and CACNA1C were not significantly associated with depression.The expressions of BDNF and CACNA1C mRNAs and proteins in adipose tissue were significantly lower in the early menopause group than those in the control group and the late menopause group,respectively,whereas the expression of ARHGEF10 was not significantly changed among the groups.Conclusion: The methylated status of BDNF and CACNA1C genes in blood could be the epigenetic markers for early menopause,and a high-PMR of BDNF gene may be one of the promising candidates for early menopause-related depression.Hypermethylation of the genes was responsible for the important epigenetic mechanism for down-expressions of BDNF and CACNA1C proteins in tissues,which affected their biological functions.