The pattern of alternative splicing and DNA methylation alteration and their interaction in linseed (Linum usitatissimum L) response to repeated drought stresses

BACKGROUND: Drought stress has significantly hampered agricultural productivity worldwide and can also result in modifications to DNA methylation levels. However, the dynamics of DNA methylation and its association with the changes in gene transcription and alternative splicing (AS) under drought stress are unknown in linseed, which is frequently cultivated in arid and semiarid regions. RESULTS: We analysed AS events and DNA methylation patterns in drought-tolerant (Z141) and drought-sensitive (NY-17) linseed under drought stress (DS) and repeated drought stress (RD) treatments. We found that the number of intron-retention (IR) and alternative 3' splice site (Alt3'SS) events were significantly higher in Z141 and NY-17 under drought stress. We found that the linseed response to the DS treatment was mainly regulated by transcription, while the response to the RD treatment was coregulated by transcription and AS. Whole genome-wide DNA methylation analysis revealed that drought stress caused an increase in the overall methylation level of linseed. Although we did not observe any correlation between differentially methylated genes (DMGs) and differentially spliced genes (DSGs) in this study, we found that the DSGs whose gene body region was hypermethylated in Z141 and hypomethylated in NY-17 were enriched in abiotic stress response Gene Ontology (GO) terms. This finding implies that gene body methylation plays an important role in AS regulation in some specific genes. CONCLUSION: Our study is the first comprehensive genome-wide analysis of the relationship between linseed methylation changes and AS under drought and repeated drought stress. Our study revealed different interaction patterns between differentially expressed genes (DEGs) and DSGs under DS and RD treatments and differences between methylation and AS regulation in drought-tolerant and drought-sensitive linseed varieties. The findings will probably be of interest in the future. Our results provide interesting insights into the association between gene expression, AS, and DNA methylation in linseed under drought stress. Differences in these associations may account for the differences in linseed drought tolerance.

DNA methylation analysis identifies key transcription factors involved in mesenchymal stem cell osteogenic differentiation

BACKGROUND: Knowledge about regulating transcription factors (TFs) for osteoblastogenesis from mesenchymal stem cells (MSCs) is limited. Therefore, we investigated the relationship between genomic regions subject to DNA-methylation changes during osteoblastogenesis and the TFs known to directly interact with these regulatory regions. RESULTS: The genome-wide DNA-methylation signature of MSCs differentiated to osteoblasts and adipocytes was determined using the Illumina HumanMethylation450 BeadChip array. During adipogenesis no CpGs passed our test for significant methylation changes. Oppositely, during osteoblastogenesis we identified 2462 differently significantly methylated CpGs (adj. p < 0.05). These resided outside of CpGs islands and were significantly enriched in enhancer regions. We confirmed the correlation between DNA-methylation and gene expression. Accordingly, we developed a bioinformatic tool to analyse differentially methylated regions and the TFs interacting with them. By overlaying our osteoblastogenesis differentially methylated regions with ENCODE TF ChIP-seq data we obtained a set of candidate TFs associated to DNA-methylation changes. Among them, ZEB1 TF was highly related with DNA-methylation. Using RNA interference, we confirmed that ZEB1, and ZEB2, played a key role in adipogenesis and osteoblastogenesis processes. For clinical relevance, ZEB1 mRNA expression in human bone samples was evaluated. This expression positively correlated with weight, body mass index, and PPARγ expression. CONCLUSIONS: In this work we describe an osteoblastogenesis-associated DNA-methylation profile and, using these data, validate a novel computational tool to identify key TFs associated to age-related disease processes. By means of this tool we identified and confirmed ZEB TFs as mediators involved in the MSCs differentiation to osteoblasts and adipocytes, and obesity-related bone adiposity.

Age Estimation Based on DNA Methylation and Its Application Prospects in Forensic Medicine / 法医学杂志

With the improvement of DNA methylation detection techniques, studies on age-related methylation sites have found more age-specific ones across tissues, which improves the sensitivity and accuracy of age estimation. In addition, the establishment of various statistical models also provides a new direction for the age estimation of tissues from different sources. This review summarizes the related studies of age estimation based on DNA methylation from the aspects of detection technology, age-related cytosine phosphate guanine site and model selection in recent years.

The critical importance of epigenetics in autoimmune-related skin diseases / 医学前沿

Autoimmune-related skin diseases are a group of disorders with diverse etiology and pathophysiology involved in autoimmunity. Genetics and environmental factors may contribute to the development of these autoimmune disorders. Although the etiology and pathogenesis of these disorders are poorly understood, environmental variables that induce aberrant epigenetic regulations may provide some insights. Epigenetics is the study of heritable mechanisms that regulate gene expression without changing DNA sequences. The most important epigenetic mechanisms are DNA methylation, histone modification, and noncoding RNAs. In this review, we discuss the most recent findings regarding the function of epigenetic mechanisms in autoimmune-related skin disorders, including systemic lupus erythematosus, bullous skin diseases, psoriasis, and systemic sclerosis. These findings will expand our understanding and highlight the possible clinical applications of precision epigenetics approaches.

Epigenetic regulation mechanism: roles in enamel formation and developmental defects of enamel / 中华口腔医学杂志

Enamel formation is a series of complex physiological processes, which are regulated by critical genes spatially and temporally. These processes involve multiple developmental stages covering ages and are prone to suffer signal interference or gene mutations, ultimately leading to developmental defects of enamel (DDE). Epigenetic modifications have important regulatory roles in gene expression during enarnel development. New technologies including high-throughput sequencing, chromatin immunoprecipitation sequencing (ChIP-seq), and DNA methylation chip are emerging in recent years, making it possible to establish genome-wide epigenetic modification profiles during developmental processes. The regulatory role of epigenetic modification with spatio-temporal pattern, such as DNA methylation, histone modification and non-coding RNA, has significantly expanded our understanding of the regulatory network of enamel formation, providing a new theoretical basis of clinical management and intervention strategy for DDE. The present review briefly describes the enamel formation process of human beings' teeth as well as rodent incisors and summarizes the dynamic characteristics of epigenetic modification during enamel formation. The functions of epigenetic modification in enamel formation and DDE are also emphatically discussed.

Research Progress on Epigenetics in Endometriosis / 中国医学科学院学报

Epigenetics refers to heritable changes in gene expression and function without alterations in gene sequences,including DNA methylation,histone modification,and non-coding RNAs.Endometriosis is a benign gynecological disease that affects the fertility and health of reproductive-age women,the etiology of which remains unclear.The recent studies have demonstrated that epigenetics plays a key role in the occurrence and development of endometriosis.This article reviews the research progress in the regulatory mechanism and application of epigenetics in endometriosis.

Advances in epigenetic regulation of Chinese hamster ovary cells / 生物工程学报

Chinese hamster ovary (CHO) cells play an irreplaceable role in biopharmaceuticals because the cells can be adapted to grow in suspension cultures and are capable of producing high quality biologics exhibiting human-like post-translational modifications. However, gene expression regulation such as transgene silencing and epigenetic modifications may reduce the recombinant protein production due to the decrease of expression stability of CHO cells. This paper summarized the role of epigenetic modifications in CHO cells, including DNA methylation, histone modification and miRNA, as well as their effects on gene expression regulation.

The chemical reprogramming of unipotent adult germ cells towards authentic pluripotency and de novo establishment of imprinting

Although somatic cells can be reprogrammed to pluripotent stem cells (PSCs) with pure chemicals, authentic pluripotency of chemically induced pluripotent stem cells (CiPSCs) has never been achieved through tetraploid complementation assay. Spontaneous reprogramming of spermatogonial stem cells (SSCs) was another non-transgenic way to obtain PSCs, but this process lacks mechanistic explanation. Here, we reconstructed the trajectory of mouse SSC reprogramming and developed a five-chemical combination, boosting the reprogramming efficiency by nearly 80- to 100-folds. More importantly, chemical induced germline-derived PSCs (5C-gPSCs), but not gPSCs and chemical induced pluripotent stem cells, had authentic pluripotency, as determined by tetraploid complementation. Mechanistically, SSCs traversed through an inverted pathway of in vivo germ cell development, exhibiting the expression signatures and DNA methylation dynamics from spermatogonia to primordial germ cells and further to epiblasts. Besides, SSC-specific imprinting control regions switched from biallelic methylated states to monoallelic methylated states by imprinting demethylation and then re-methylation on one of the two alleles in 5C-gPSCs, which was apparently distinct with the imprinting reprogramming in vivo as DNA methylation simultaneously occurred on both alleles. Our work sheds light on the unique regulatory network underpinning SSC reprogramming, providing insights to understand generic mechanisms for cell-fate decision and epigenetic-related disorders in regenerative medicine.

Genome-wide DNA methylation and transcriptome expression profiles of peripheral blood mononuclear cells in patients with systemic sclerosis with interstitial lung disease / 中南大学学报(医学版)

OBJECTIVES@#This study aims to investigate the genome-wide DNA methylation and transcriptome expression profiles of peripheral blood mononuclear cells (PBMCs) in patients with systemic sclerosis (SSc) with interstitial lung disease (ILD), and to analyze the effects of DNA methylation on Wnt/β-catenin and chemokine signaling pathways.@*METHODS@#PBMCs were collected from 19 patients with SSc (SSc group) and 18 healthy persons (control group). Among SSc patients, there were 10 patients with ILD (SSc with ILD subgroup) and 9 patients without ILD (SSc without ILD subgroup). The genome-wide DNA methylation and gene expression level were analyzed by using Illumina 450K methylation chip and Illumina HT-12 v4.0 gene expression profiling chip. The effect of DNA methylation on Wnt/β-catenin and chemokine signal pathways was investigated.@*RESULTS@#Genome-wide DNA methylation analysis identified 71 hypermethylated CpG sites and 98 hypomethylated CpG sites in the SSc with ILD subgroup compared with the SSc without ILD subgroup. Transcriptome analysis distinguished 164 upregulated genes and 191 downregulated genes in the SSc with ILD subgroup as compared with the SSc without ILD subgroup. In PBMCs of the SSc group, 35 genes in Wnt/β-catenin signaling pathway were hypomethylated, while frizzled-1 (FZD1), mitogen-activated protein kinase 9 (MAPK9), mothers against DPP homolog 2 (SMAD2), transcription factor 7-like 2 (TCF7L2), and wingless-type MMTV integration site family, member 5B (WNT5B) mRNA expressions were upregulated as compared with the control group (all P<0.05). Compared with the SSc without ILD subgroup, the mRNA expressions of dickkopf homolog 2 (DKK2), FZD1, MAPK9 were upregulated in the SSc with ILD subgroup, but the differences were not statistically significant (all P>0.05). In PBMCs of the SSc group, 38 genes in chemokine signaling pathway were hypomethylated, while β-arrestin 1 (ARRB1), C-X-C motif chemokine ligand 10 (CXCL10), C-X-C motif chemokine ligand 16 (CXCL16), FGR, and neutrophil cytosolic factor 1C (NCF1C) mRNA expressions were upregulated as compared with the control group (all P<0.05). Compared with the SSc without ILD subgroup, the mRNA expressions of ARRB1, CXCL10, CXCL16 were upregulated in the SSc with ILD subgroup, but the differences were not statistically significant (all P>0.05).@*CONCLUSIONS@#There are differences in DNA methylation and transcriptome profiles between SSc with ILD and SSc without ILD. The expression levels of multiple genes in Wnt/β- catenin and chemokine signaling pathways are upregulated, which might be associatea with the pathogenesis of SSc.

Research Progress on Gene Mutation and CHIP in Pathogenesis of MDS --Review / 中国实验血液学杂志

With the development of molecular biology techniques, the people's understanding of myelodysplastic syndromes (MDS) has greatly improved, a heterogeneous hematopoietic pre-malignant disorder of the stem cells. Gene mutations include RNA splicing, DNA methylation, chromosome modification, transcription factors, signal transduction kinases, RAS pathways, cohesion complexes, DNA repair, etc. Gene mutation is the determinant of diagnostic typing and therapeutic efficacy of MDS. The new concepts of CHIP and ICUS have aroused people's attention to the elderly patients with clonal hematopoiesis and non-clonal cytopenia but without MDS characteristics, who have the possibility of high-risk transformation to MDS and leukemia. In order to better understand the pathogenesis of MDS, the significance of gene mutations, CHIP and ICUS in the diagnosis and prognosis of MDS were reviewed in this paper.

Clinical Significance of SFRP1 Gene Methylation in Patients with Childhood Acute Lymphoblastic Leukemia / 中国实验血液学杂志

OBJECTIVE@#To investigate the clinical significance of SFRP1 gene and its methylation in childhood acute lymphoblastic leukemia (ALL) .@*METHODS@#Methylation-specific PCR (MSP) was used to detect the methylation status of SFRP1 gene in bone marrow mononuclear cells of 43 children with newly diagnosed ALL before chemotherapy (primary group) and when the bone marrow reached complete remission d 46 after induction of remission chemotherapy (remission group), the expression of SFRP1 mRNA was detected by quantitative real-time polymerase chain reaction (qRT-PCR), the expression of SFRP1 protein was detected by Western blot, and clinical data of children were collected, the clinical significance of SFRP1 gene methylation in children with ALL was analyze.@*RESULTS@#The positive rate of SFRP1 gene promoter methylation in the primary group (44.19%) was significantly higher than that in the remission group (11.63%) (χ2=11.328, P<0.05). The relative expression levels of SFRP1 mRNA and protein in bone marrow mononuclear cells of children in the primary group were significantly lower than those in the remission group (P<0.05). Promoter methylation of SFRP1 gene was associated with risk level (χ2=15.613, P=0.000) and survival of children (χ2=6.561, P=0.010) in the primary group, children with SFRP1 hypermethylation had significantly increased risk and shortened event-free survival time, but no significant difference in other clinical data.@*CONCLUSION@#Hypermethylation of SFRP1 gene promoter may be involved in the development of childhood ALL, and its hypermethylation may be associated with poor prognosis.

Association Analysis Between Methylation of SCARB1 Gene Promoter and Coronary Heart Disease / 中国医学科学院学报

Objective To explore the relationship between scavenger receptor class B member 1 (SCARB1) gene promoter methylation and the pathogenesis of coronary artery disease. Methods A total of 120 patients with coronary heart disease treated in Renji Hospital affiliated to Shanghai Jiao Tong University School of Medicine from December 2018 to May 2020 were selected as the case group,while 140 gender and age matched healthy participants were randomly selected as the control group for a case-control study.The methylation status was detected by high-throughput target sequencing after bisulfite converting,and the methylation of CpG sites in the promoter region of SCARB1 gene was compared between the two groups. Results The case group showed higher methylation level of SCARB1+67 and lower methylation level of SCARB1+134 than the control group (both P<0.001),and the differences remained statistically significant in men (both P<0.001) and women (both P<0.001).The overall methylation level in the case group was lower than that in the control group [(80.27±2.14)% vs.(81.11±1.27)%;P=0.006],while this trend was statistically significant only in men (P=0.002). Conclusion The methylation of SCARB1 gene promotor is associated with the pathogenesis and may participate in the occurrence and development of coronary heart disease.

Research progress on the role and clinical significance of DNA methylation in early nutritional programming / 生理学报

Early life nutritional environment is not only associated with the growth and development of children, but also affects the health of adults. Numerous epidemiological and animal studies suggest that early nutritional programming is an important physiological and pathological mechanism. DNA methylation is one of the important mechanisms of nutritional programming, which is catalyzed by DNA methyltransferase, a specific base of DNA covalently binds to a methyl group, to regulate gene expression. In this review, we summarize the role of DNA methylation in the "abnormal developmental planning" of key metabolic organs caused by excessive nutrition in early life, resulting in long-term obesity and metabolic disorders in the offspring, and explore the clinical significance of regulating DNA methylation levels through dietary interventions to prevent or reverse the occurrence of metabolic disorders in the early stage in a "deprogramming" manner.

Research Progress of DNA Methylation in Cisplatin Resistance in Lung Cancer / 中国肺癌杂志

As one of the most common malignant tumors, lung cancer poses a serious threat to human life and health. The platinum-based drug cisplatin (DDP) is used as the first-line treatment for lung cancer. The poor prognosis of lung cancer is mostly due to developed resistance to cisplatin, which poses a serious treatment challenge. The mechanism of cisplatin resistance is complex and unclear. Numerous studies have shown that DNA methylation plays a crucial role in the emergence of lung cancer cisplatin resistance. DNA hypermethylation results in the deactivation of numerous drug resistance genes and tumor suppressor genes through a change in chromatin conformation. Finding new therapeutic targets and indicators to predict the therapeutic effect can be aided by elucidating the complex mechanism. In order to discover novel strategies to overcome cisplatin resistance in lung cancer, this paper discusses DNA methylation-mediated cisplatin resistance and offers an overview of current demethylation procedures.
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Influence of E-cadherin methylation on prognosis in children with acute lymphoblastic leukemia / 中国当代儿科杂志

OBJECTIVES@#To study the significance of E-cadherin and the association between E-cadherin methylation status and prognosis in children with acute lymphoblastic leukemia (ALL) by examining the mRNA and protein expression of E-cadherin and its gene methylation status in bone marrow mononuclear cells of children with ALL.@*METHODS@#The samples of 5 mL bone marrow blood were collected from 42 children with ALL who were diagnosed for the first time at diagnosis (pre-treatment group) and on day 33 of induction chemotherapy (post-treatment group). RT-qPCR, Western blot, and methylation-specific PCR were used to measure the mRNA and protein expression of E-cadherin and the methylation level of the E-cadherin gene. The changes in each index after induction chemotherapy were compared.@*RESULTS@#The mRNA and protein expression levels of E-cadherin in the post-treatment group were significantly higher than those in the pre-treatment group (P<0.05), while the positive rate of E-cadherin gene methylation in the post-treatment group was significantly lower than that in the pre-treatment group (P<0.05). At the end of the test, the children with negative methylation had significantly higher overall survival rate and event-free survival rate than those with positive methylation (P<0.05).@*CONCLUSIONS@#E-cadherin expression is associated with the development of ALL in children, and its decreased expression and increased methylation level may indicate a poor prognosis.

Perfil da metilação do DNA do binômio mãe-filho no contexto da obesidade materna, adiposidade fetal e neonatal / DNA methylation profile of the mother-child binomial in maternal obesity, fetal and neonatal adiposity

Introdução: Um mecanismo epigenético pelo qual os efeitos adversos do ambiente intra-uterino são transferidos aos descendentes é a metilação do DNA. Objetivo: Avaliar a relação entre obesidade materna, ganho de peso gestacional (GPG) e alteração da metilação do DNA sobre o desenvolvimento fetal e neonatal. Metodologia: Subpopulação de gestantes do estudo epidemiológico prospectivo "Coorte Araraquara" foram acompanhadas durante os três trimestres da gestação, parto e no pós-parto. Para avaliar o impacto do GPG na metilação do DNA e desenvolvimento fetal e neonatal, as gestantes foram alocadas em 2 grupos: ganho de peso gestacional adequado (n=45) e ganho de peso gestacional excessivo (n=30). Para avaliar o impacto da obesidade materna na metilação do DNA e desenvolvimento fetal e neonatal, as gestantes foram alocadas em 2 grupos: A. IMC pré-gestacional adequado (n=25) e B. IMC pré-gestacional sobrepeso/obesidade (n=39 gestantes). A biometria e composição corporal fetal foram avaliadas por ultrassom Siemens ACUSON X300TM (Siemens®, Mountain View, CA, USA) e a composição corporal do neonato por pletismografia, com o PEA POD (Cosmed®, Concord, CA, USA). A dieta materna ao final da gestação foi investigada por recordatórios de 24 horas e analisada no software Nutrition Data System for Research (NDSR, Minnesota, USA). O DNA dos sangues materno e cordão umbilical, da vilosidade e decídua placentárias foram extraídos utilizando proteinase K pelo método santing-out. As regiões hipo e hipermetiladas foram analisados pela técnica de Digestão Enzimática Sensível à Metilação associada à PCR quantitativa (qPCR) e a expressão gênica por qPCR. Para análise da influência do GPG o DNA do sangue materno de gestantes com GPG adequado (N=8) versus com GPG excessivo (N=8), foi hibridizado na plataforma Illumina com o Human Methylation 850K Bead Chip (Illumina, CA, USA). Para análise estatística aplicou-se o Teste t, Qui-quadrado (X2), ANOVA de medidas repetidas e modelos de regressão linear múltiplo e o nível de significância adotado foi de p≤0,05. Resultados: O excessivo ganho de peso gestacional (EGPG) alterou os triglicerídeos (TGs) e colesterol total (CT) maternos, o diâmetro occipito-frontal (DOF) do feto, a circunferência da cabeça, perímetro torácico, peso e a massa gorda neonatais. A análise de metilação global do DNA materno identificou 46 posições diferencialmente metiladas e 11 regiões diferencialmente metiladas (DMRs). Nove fenótipos humanos foram enriquecidos para essas 11 DMRs localizadas em 13 genes (EMILIN1, HOXA5, CPT1B, CLDN9, ZFP57, BRCA1, POU5F1, ANKRD33, HLA-B, RANBP17, ZMYND11, DIP2C, TMEM232), destacando-se os termos resistência à insulina e hiperglicemia. O DNAm materno foi associado com parâmetros de composição corporal, como: tecido total da coxa e do braço fetais e gordura subcutânea da coxa e do braço fetais, bem como ao percentual de massa gorda e massa gorda neonatais. A metilação do DNA materno também foi associada com os TGs e a insulina de jejum maternos, com circunferência abdominal e circunferência da cabeça fetais (CC) e CC neonatal. As DMRs estudadas foram enriquecidas em 142 processos biológicos, 21 funções moleculares e 17 componentes celulares. Três módulos gênicos diferencialmente metilados foram identificados. Por outro lado, o IMC pré-gestacional sopreso/obesidade alterou os percentis do diâmetro biparietal (DBF), o DOF, a CC, a espessura da gordura subcutânea do abdômen (ETSA), tecido total do braço, massa muscular do braço e gordura subcutânea do braço fetais e a CC do neonato. O gene H19DMR foi significativamente menos metilado no sangue materno do grupo com sobrepeso/obesidade em comparação ao grupo com IMC pré-gestacional adequado, para o sangue do cordão umbilical e tecidos placentários, não houve diferença de metilação entre os grupos, bem como não houve diferença de expressão gênica dos genes H19 e IGF2 nos tecidos placentários entre os grupos. Houve associações entre metilação de H19DMR no sangue do cordão umbilical com os percentis do DBP, com a ETSA e a CC neonatais; na decídua com o DOF, a CC e comprimento fetais; no vilo com o DOF, a CC e a ETSA fetais e com a CC neonatal. A expressão do gene H19 na decídua também foi associada ao DBP e ao percentil do comprimento do fêmur fetais; no vilo com o DOF e gordura subcutânea do braço fetais. A expressão do gene IGF2 na decídua foi associada com o DBP fetal e no vilo com o DOF fetal. Conclusão: A metilação do DNA foi alterada pelo ganho de peso gestacional e obesidade maternos e se associou com a adiposidade e crescimento fetais. O excessivo GPG alterou o metiloma materno e está envolvido com um fenótipo de risco para o desenvolvimento de doenças crônicas e metabólicas, a exemplo da diabetes.

Introduction: An epigenetic mechanism by which the adverse effects of the intrauterine environment are transferred to offspring is DNA methylation. Objective: Evaluate the relationship between maternal obesity, gestational weight gain and DNA methylation alteration on fetal and neonatal development. Methodology: Subpopulation of pregnant women from the prospective epidemiological study "Cohorte Araraquara" were followed during the three trimesters of pregnancy, delivery and postpartum and were allocated into 2 groups: A. adequate weight (N=25) and B. overweight/obesity (N=39 pregnant women). Fetal biometry and adiposity were evaluated by ultrasound and the neonate's body composition by plethysmography, with the PEA POD (Cosmed®, Concord, CA, USA). Maternal diet in the end of pregnancy was investigated using 24-hour recalls and analyzed using the Nutrition Data System for Research software (NDSR, Minnesota, USA). DNA maternal and umbilical cord blood, and placental villus and decidua were extracted using proteinase K by the santing-out method. The hypo and hypermethylated regions were analyzed by the Methylation Sensitive Enzymatic Digestion technique associated with quantitative PCR (qPCR) and gene expression by qPCR. To analyze the influence of gestational weight gain (GWG), maternal blood DNA from pregnant women with adequate AGWG (N=8) versus excessive EGWG (N=8) was hybridized on the Human Methylation 850K Bead Chip (Illumina, CA). For statistical analysis, the t test, chi-square (X2) test, repeated measures ANOVA and multiple linear regression models were applied, and the significance level adopted was p≤0.05. Results: The pre-gestational weight, pre-gestational BMI and BMI during pregnancy were higher in group B. In this same group, higher values of fasting insulin, us-CRP and lipid consumption were found in relation to group A. subcutaneous abdominal fat thickness (SCFT) and subcutaneous fat of the fetal arm was also higher in group B compared to group A. Regarding the body composition of neonates, the amount of fat mass was higher in group B. The H19DMR gene was less methylated in maternal blood from group B. No statistical difference was found in the values of IGF2 and H19 gene expression between the groups. There were associations between methylation of the H19DMR gene in umbilical cord blood with fetal biparietal diameter (BPD) and SCFT and neonatal head circumference (HC); in the decidua with occipito frontal diameter (OFD), fetal length and HC; in the villi with DOF, HC and SCFT and neonatal HC. Excessive gestational weight gain altered 46 CpG sites, 11 differentially methylated regions (DMRs) located in 13 genes, namely: EMILIN1, HOXA5, CPT1B, CLDN9, ZFP57, BRCA1, POU5F1, ANKRD33, HLA-B, RANBP17, ZMYND11, DIP2C, TMEM232. These DMRs were enriched in 142 biological processes, 21 molecular functions, 17 cellular components and 9 human phenotypes. In addition, 3 differentially methylated gene modules were identified to the phenotype of interest. Conclusion: DNA methylation was altered by maternal nutritional status and was associated with fetal adiposity and growth. Excessive GPG altered maternal methylome and was associated with the phenotype of metabolic diseases such as diabetes mellitus.

Estudo piloto de triagem usando a Análise da Curva de Dissociação de Alta Resolução Sensível à Metilação (MS -HRM) em swabs orais para detectar a Síndrome de Prader-Willi / Pilot screening study using Methylation Sensitive High Resolution Dissociation Curve Analysis (MS -HRM) on oral swabs to detect Prader-Willi Syndrome

A síndrome de Prader-Willi (SPW) é uma desordem genética complexa, caracterizada por deleções, dissomia uniparental materna ou defeito no centro de imprinting no alelo paterno do cromossomo 15. As perdas de funções de genes específicos da região 15q11 afetam múltiplos sistemas corporais. O diagnóstico da SPW é difícil de ser realizado com base apenas no exame clínico e envolve a realização de diversas técnicas de biologia molecular para a completa elucidação da etiologia genética, tornando todo o processo laborioso, demorado e custoso. A realização de um teste molecular que permita um diagnóstico rápido e preciso é de vital importância para um melhor prognóstico para paciente. A coleta bem-sucedida de amostras e a extração de DNA de swabs são alternativas não invasivas e confiáveis, tanto para os pacientes quanto para os profissionais que realizarão a coleta destas amostras. Neste trabalho foi possível demonstrar um método simples de coleta de amostras e extração de DNA, que possui baixo custo, é eficaz, fácil e rápido, que fornece uma quantidade e qualidade suficiente de DNA para a execução do MS-HRM, qPCR e sequenciamento. Uma comparação dos procedimentos de extração mostra que o método simples de extração de NaCl é o mais adequado para extração de DNA de amostra bucal coletada através de swab. Neste trabalho foi demonstrado um método simples de coleta de amostras através do swab e extração de DNA com baixo custo e boa qualidade do DNA.

Prader-Willi syndrome (PWS) is a complex disorder, uniparental by deletions, dissociated from no imprint defect in any of the chromosomes 15. As gene variants of the genetic region of the 15q11 region, the diagnosis of PWS is challenging to perform based on clinical examination alone. It involves the performance of several molecular biology techniques for the complete elucidation of genetics, determining the entire laborious, time-consuming, and costly process. The performance of a molecular test allows a quick diagnosis, which is vital for a better prognosis for the patient. Successful sample collection and DNA collection from swabs are non-invasive alternatives for patients and practitioners performing probable sample collection. In this work, it was possible to demonstrate a simple sample collection and DNA method, which has a low cost, is effective, easy, and fast, and provides a sufficient quantity and quality of DNA for the execution of MS-HRM, qPCR, and sequencing. A comparison of the extraction procedures shows that the simple NaCl extraction method is the most appropriate for extracting DNA from a buccal sample collected via swab. In this work, a simple method for swab sampling and extracting DNA was demonstrated, with low cost and good DNA quality.

Chinese Medicine Regulates DNA Methylation to Treat Haematological Malignancies: A New Paradigm of "State-Target Medicine" / 中国结合医学杂志

Aberrant regulation of DNA methylation plays a crucial causative role in haematological malignancies (HMs). Targeted therapy, aiming for DNA methylation, is an effective mainstay of modern medicine; however, many issues remain to be addressed. The progress of epigenetic studies and the proposed theory of "state-target medicine" have provided conditions to form a new treatment paradigm that combines the "body state adjustment" of CM with targeted therapy. We discussed the correlation between Chinese medicine (CM) syndromes/states and DNA methylation in this paper. Additionally, the latest research findings on the intervention and regulation of DNA methylation in HMs, including the core targets, therapy status, CM compounds and active components of the Chinese materia medica were concisely summarized to establish a theoretical foundation of "state-target synchronous conditioning" pattern of integrative medicine for HMs, simultaneously leading a new perspective in clinical diagnosis and therapy.

DNA Methylation and Transcription of HLA-F and Serum Cytokines Relate to Chinese Medicine Syndrome Classification in Patients with Chronic Hepatitis B / 中国结合医学杂志

OBJECTIVE@#To explore the molecular bases of Chinese medicine (CM) syndrome classification in chronic hepatitis B (CHB) patients in terms of DNA methylation, transcription and cytokines.@*METHODS@#Genome-wide DNA methylation and 48 serum cytokines were detected in CHB patients (DNA methylation: 15 cases; serum cytokines: 62 cases) with different CM syndromes, including dampness and heat of Gan (Liver) and gallbladder (CHB1, DNA methylation: 5 cases, serum cytokines: 15 cases), Gan stagnation and Pi (Spleen) deficiency (CHB2, DNA methylation: 5 cases, serum cytokines: 15 cases), Gan and Shen (Kidney) yin deficiency (CHB3, DNA methylation: 5 cases, serum cytokines: 16 cases), CHB with hidden symptoms (HS, serum cytokines:16 cases) and healthy controls (DNA methylation: 6 cases). DNA methylation of a critical gene was further validated and its mRNA expression was detected on enlarged samples. Genome-wide DNA methylation was detected using Human Methylation 450K Assay and furthered verified using pyrosequencing. Cytokines and mRNA expression of gene were evaluated using multiplex biometric enzyme-linked immunosorbent assay (ELISA)-based immunoassay and reverse transcription-quantitative polymerase chain reaction (RT-qPCR), respectively.@*RESULTS@#Totally 28,667 loci, covering 18,403 genes were differently methylated among CHB1, CHB2 and CHB3 (P<0.05 and |Δβ value| > 0.17). Further validation showed that compared with HS, the hg19 CHR6: 29691140 and its closely surrounded 2 CpG loci were demethylated and its mRNA expressions were significantly up-regulated in CHB1 (P<0.05). However, they remained unaltered in CHB2 (P>0.05). Levels of Interleukin (IL)-12 were higher in CHB3 and HS than that in CHB1 and CHB2 groups (P<0.05). Levels of macrophage inflammatory protein (MIP)-1α and MIP-1β were higher in CHB3 than other groups and leukemia inhibitory factor level was higher in CHB1 and HS than CHB2 and CHB3 groups (P<0.05). IL-12, MIP-1α and MIP-1β concentrations were positively correlated with human leukocyte antigen F (HLA-F) mRNA expression (R2=0.238, P<0.05; R2=0.224, P<0.05; R=0.447, P<0.01; respectively). Furthermore, combination of HLA-F mRNA and differential cytokines greatly improved the differentiating accuracy among CHB1, CHB2 and HS.@*CONCLUSIONS@#Demethylation of CpG loci in 5' UTR of HLA-F may up-regulate its mRNA expression and HLA-F expression was associated with IL-12, MIP-1α and MIP-1β levels, indicating that HLA-F and the differential cytokines might jointly involve in the classification of CM syndromes in CHB.@*REGISTRATION NO@#ChiCTR-RCS-13004001.

Effect of miR-203/CREB1 Signaling Regulation Mediated by DNA Methylation on the Proliferation and Apoptosis of Multiple Myeloma Cells / 中国实验血液学杂志

OBJECTIVE@#To investigate the effect of miR-203/CREB1 signaling regulation mediated by DNA methylation on the proliferation, invasion and apoptosis of multiple myeloma (MM) cells.@*METHODS@#The methylation level of miR-203 in the RPMI 8226 cells was detected by bisulfite sequcucing polymerase chain reaction (BSP). The mRNA expression of miR-203 was measured by quantitative real-time polymerase chain reaction. RPMI 8226 cells were treated with DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine (5-Aza-CdR). The miR-203 mimic in MM cell line RPMI 8226 was transfected to establish overexpressed miR-203 cell. The proliferation, invasion ability and apoptosis of RPMI 8226 cell was detected by CCK-8 assay, Transwell, and flow cytometry, respectively. The targeting relationship between miR-203 and CREB1 was verified by double luciferase report assay. Western blot was used to detect the expression of CREB1 protein.@*RESULTS@#Hypermethylation of miR-203 promoter region and low expression level of miR-203 mRNA were detected in the RPMI 8226 cells, which showed that demethylation could induce the expression of miR-203. The proliferation and invasion ability of RPMI 8226 cells after treated by 5-Aza-CdR were inhibited, and showed statistical significance as compared with blank control group (both P<0.05)，while the apoptosis rate was promoted (P<0.05). The proliferation, invasion ability and apoptosis of overexpressed miR-203 were the same as the demethylation group. Double luciferase report assay confirmed that CREB1 was the direct target of miR-203. The protein level of CREB1 was inhibited by demethylation and showed statistical significance as compared with control group (P<0.05).@*CONCLUSION@#MiR-203 targeting CREB1 mediated by DNA methylation leads to maintain the malignant biological behaviors of MM cells.