RESUMEN
Objective:To investigate the effects of chronic stress during pregnancy on depressive behavior and DNA methylation of insulin-like growth factor-2 ( IGF-2 )/long non-coding RNA ( lncRNA ) H19 in hippocampus of female offspring rats.Methods:A total of 32 SPF female SD rats were divided into model group and control group according to the random number table. The rats in the model group were treated with chronic unpredictable mild stress (CUMS) to establish the depression model, and the rats in the control group were fed normally.On the 7th day of stress stimulation, all female rats mated with male rats. One day before stress stimulation and 1, 7, 14, 21, 28 days after stress stimulation, blood samples were collected from the inner canthus vein of the rats to determine the plasma corticosterone concentration. Eight female pups were randomly selected from each group on postnatal day 28(PND28) and postnatal day 42 (PND42). Plasma corticosterone concentration was measured after angular vein blood collection. At PND42, the depression-like behavior of female pups in the two groups was measured by sucrose preference test, tail suspension test and forced swimming test. The expression of IGF-2/H19 and related transferases in hippocampus of offspring rats was detected by RT-PCR and Western blot. Methyl target technology was used to capture and sequence 19 CpG sites of IGF-2 differentially methylated region(DMR) fragment 2, 8 CpG sites in H19 imprinting control region (ICR) fragment 1 and 15 CpG sites in H19-ICR fragment 2, and calculate the methylation level of each CpG site. SPSS 26.0 was used for statistical analysis of relevant data by repeated measurement ANOVA, t test and non-parametric test. Results:(1) The data of plasma corticosterone content of the two groups of female rats at different times were analyzed by repeated measurement variance.The results showed that the the interaction effect between time and group was not significant ( F=2.997, P=0.066), and the main effect of time was significant ( F=4.44, P=0.010). The main effect of group was significant ( F=41.40, P=0.001). According to the independent effect analysis of factors between groups, on the 14th, 21st, and 28th days of stress, the plasma corticosterone concentration of the model group was higher than that of the control group (all P<0.001). (2) In the sucrose preference test, the total liquid consumption (11.10(10.38, 11.58) mL, 13.55(12.00, 15.77) mL, Z=-3.055, P=0.002), 1% sucrose water consumption ((5.50±1.30) mL, (8.56±2.04) mL, t=-3.582, P=0.003) and 1% sucrose preference percentage ( (51.35±8.69) %, (62.11±8.05) %, t=-2.576, P=0.022) of female pups in the model group were significantly lower than those in the control group. (3) The duration of immobility in tail suspension test ((126.95±39.89) s, (54.30±25.00) s, t=4.375, P=0.001) and forced swimming test ((7.97±6.66) s, (1.85±2.12) s, t=2.478, P=0.037) of female offspring in the model group were longer than those in the control group. (4) The expression of IGF-2 mRNA ((0.46±0.24), (1.00±0.00), t=3.821, P=0.019) and H19 mRNA ((0.60±0.25), (1.00±0.00), t=3.574, P=0.007) in hippocampus of female pups in the model group were lower than those of control group. The relative expression of IGF-2 protein in female offspring of model group was lower than that in control group ((0.77±0.04), (1.00±0.00), t=9.876, P=0.01). The relative expression of CCTC-binding factor (CTCF) mRNA ((1.29±0.12), (1.00±0.00), t=-4.850, P=0.003) and protein ((1.90±0.28), (1.00±0.00), t=-5.513, P=0.005) were higher than those in the control group. (5) The methylation levels of three CpG sites in the IGF-2 DMR region of female offspring in the model group were lower than those in the control group ( t=-3.21, -3.00, -3.34, all P<0.05), located at chr1215831028, chr1215831055 and chr1215831205, respectively. The methylation level of IGF-2 DMR fragment was lower than that of the control group ( t=-3.453, P=0.048). The relative expression levels of DNMT3A mRNA ( t=5.102, P=0.002), DNMT3A ( t=10.213, P<0.001) and DNMT3B ( t=4.169, P=0.014) in female offspring of the model group were lower than those in the control group. Conclusion:Chronic stress during pregnancy causes depression and despair in female offspring mice, and the mechanism may be related to the decrease of methylation level of imprinted gene IGF-2 DMR caused by the decrease of methyltransferase expression.
RESUMEN
Breast cancer is an important public health problem. As mammary gland development is a dynamic process that initiates in embryonic life, recent evidence show that in-utero life exposure to maternal nutritional factors can alter mammary gland development and program breast cancer risk in adult life. Even tough studies focus on maternal nutrition, recent evidence show that paternal nutritional factors in-utero and during preconception also affects their female offspring mammary gland development and breast cancer susceptibility in adult life. Studies highlight epigenetic modulation of gene expression in the mammary gland as possible breast cancer programming underlying mechanisms. Selenium is a micronutrient with essential role in central aspects of embryogenesis, male fertility and that has been extensively studied as a chemopreventive agent in several breast cancer models. Among selenium possible mechanisms of action, modulation of cell proliferation, apoptosis, DNA damage, gene expression and epigenetic marks are highlighted. Thus, a rat experiment was conducted to evaluate whether paternal selenium deficiency or supplementation during preconception could affect mammary gland development and breast cancer risk, as well as possible molecular mechanisms involved. Four-week old male Sprague-Dawley rats were exposed to experimental diets (AIN93G) containing 0.15 (control), 0.05 (deficient) and 1ppm (supplemented) of selenium as sodium selenate for 9 weeks and mated with control females. At 7-week old, mammary carcinogenesis was induced in their female offspring by oral administration of 7,12 dymethylbenz[a] anthracene and mammary neoplasia development was evaluated. Paternal selenium deficiency during preconception altered mammary gland development as increased terminal end buds (TEBs) number, epithelial elongation and cell proliferation and decreased apoptosis that were associated with increased breast cancer risk (higher incidence and grade tumors). In addition, paternal selenium deficiency during preconception induced molecular alterations in the mammary gland of the female offspring such as global DNA hypomethylation, increased global levels of H3K27me3 and altered expression of genes related to early life and mammary gland development, apoptosis, cell cycle control, and DNA damage repair. Paternal selenium supplementation during preconception on the other hand did not influence breast cancer programing. Our data show that breast cancer risk can be determined in early-life stages trough the male germline molecular modulation and preconception as an important window of opportunity to start breast cancer prevention strategies. Assuring and adequate selenium intake by men could be a possible starting point
O câncer de mama é um importante problema de saúde pública. O desenvolvimento da glândula mamária é um processo dinâmico que se inicia na vida intrauterina e evidências recentes mostram que a exposição do feto a fatores nutricionais maternos altera o desenvolvimento da glândula mamária e a susceptibilidade ao câncer de mama na vida adulta. Mesmo com um maior foco na nutrição materna, evidências recentes apontam que a nutrição paterna no período intrauterino e de preconcepção também afetam o desenvolvimento da glândula mamária e o risco de câncer de mama da sua prole feminina na vida adulta. Estudos apontam a modulação epigenética da expressão de genes na glândula mamária como possíveis mecanismos envolvidos na programação do câncer de mama. O selênio é um micronutriente com papel essencial em aspectos centrais da embriogênese, fertilidade masculina e que tem sido extensivamente estudado como um agente quimiopreventivo em diferentes modelos de câncer de mama. Dentre os possíveis mecanismos de ação do selênio, destacam-se a capacidade de modulação da proliferação celular, apoptose, danos do DNA e da expressão de genes e mecanismos epigenéticos. Dessa forma, foi conduzido um experimento em ratos para avaliar se a deficiência ou suplementação paterna com selênio durante o período de preconcepção poderia afetar na prole feminina o desenvolvimento da glândula mamária e o risco ao câncer de mama na vida adulta, assim como possíveis mecanismos moleculares envolvidos. Ratos machos da linhagem Sprague-Dawley com 4 semanas de vida foram submetidos à dieta experimental AIN93G contendo 0,15 (controle); 0,05 (deficiente) e 1ppm (suplementada) com selênio na forma de selenato de sódio por 9 semanas e acasalados com fêmeas controle. Com 7 semanas de vida, a carcinogênese mamária foi iniciada na prole feminina através da administração oral do carcinógeno químico 7,12 dimetilbenz[a] antraceno e o desenvolvimento das neoplasias mamárias foi avaliado. A deficiência paterna de selênio causou alterações no desenvolvimento da glândula mamária da prole feminina como aumento no número de terminal end buds (TEBs), aumento da elongação do epitélio mamário, aumento da proliferação celular e diminuição da apoptose que foram associados ao aumento do risco do câncer de mama (maior incidência e agressividade das lesões). Além disso, a deficiência paterna de selênio causou alterações de nível molecular na glândula mamária da prole feminina como hipometilação global, aumento dos níveis globais de H3K27me3 e alteração na expressão de genes relacionados ao desenvolvimento no início da vida e da glândula mamária, apoptose, controle de ciclo celular e reparo de danos no DNA. A suplementação paterna com selênio não foi influenciou o desenvolvimento da glândula mamária e o risco ao câncer de mama na vida adulta. Nossos resultados mostram que o risco do câncer de mama pode ser determinado no início da vida através de influências paternas por meio da modulação de mecanismos moleculares e que o período de preconcepção se caracteriza como uma importante janela de susceptibilidade para iniciar estratégias de diminuição do risco do câncer de mama. Assegurar uma ingestão adequada de selênio por homens pode ser um possível ponto de partida