O6-methylguanine DNA methyltransferase(MGMT), a DNA repair enzyme, has been reported in some congenital malformations, but it is less frequently reported in neural tube defects. This study investigated MGMT mRNA expre...O6-methylguanine DNA methyltransferase(MGMT), a DNA repair enzyme, has been reported in some congenital malformations, but it is less frequently reported in neural tube defects. This study investigated MGMT mRNA expression and methylation levels in the early embryo and in different embryonic stages, as well as the relationship between MGMT and neural tube defects. Spina bifida aperta was induced in rats by a single intragastric administration of all-trans retinoic acid on embryonic day(E) 10, whereas normal control rats received the same amount of olive oil on the same embryonic day. DNA damage was assessed by detecting γ-H2 A.X in spina bifida aperta rats. Real time-polymerase chain reaction was used to examine mRNA expression of MGMT in normal control and spina bifida aperta rats. In normal controls, the MGMT mRNA expression decreased with increasing embryonic days, and was remarkably reduced from E11 to E14, reaching a minimum at E18. In the spina bifida aperta model, γ-H2 A.X protein expression was increased, and mRNA expression of MGMT was markedly decreased on E14, E16, and E18. Bisulfite sequencing polymerase chain reaction for MGMT promoter methylation demonstrated that almost all CpG sites in the MGMT promoter remained unmethylated in both spina bifida aperta rats and normal controls, and there was no significant difference in methylation level between the two groups on either E14 or E18. Our results show that DNA damage occurs in spina bifida aperta rats. The mRNA expression of MGMT is downregulated, and this downregulation is independent of promoter DNA methylation.展开更多
N^(6)-methyladenosine RNA methylation,an essential post-transcriptional modification,dynamically regulates RNA metabolism and plays a crucial role in neuronal function.Growing evidence suggests that dysregulated N^(6)...N^(6)-methyladenosine RNA methylation,an essential post-transcriptional modification,dynamically regulates RNA metabolism and plays a crucial role in neuronal function.Growing evidence suggests that dysregulated N^(6)-methyladenosine modification contributes to the pathogenesis of neurodegenerative diseases,including Alzheimer’s disease,Parkinson’s disease,multiple sclerosis,and amyotrophic lateral sclerosis.However,the precise mechanisms by which N^(6)-methyladenosine modification influences these conditions remain unclear.This review summarizes the role of m6A modification and its associated regulators in neurodegeneration,focusing on their involvement in key pathological processes.In Alzheimer’s disease,m6A modification contributes to synaptic dysfunction,mitochondrial damage,and neuronal apoptosis.Evidence from APP/PS1,5xFAD,tau transgenic,and Drosophila models demonstrates that regulators such as methyltransferase-like 3 and fat mass and obesity-associated protein influence Alzheimer’s disease progression through neuroinflammation,circular RNAs dysregulation,and autophagy-related mechanisms.In Parkinson’s disease,altered N^(6)-methyladenosine regulator expression affects dopaminergic neuron survival and stress responses by modulating mRNA stability and autophagy-related lncRNAs.In multiple sclerosis and amyotrophic lateral sclerosis,N^(6)-methyladenosine affects immune activation,myelin repair,and the regulation of disease-associated genes such as TDP-43.Beyond N^(6)-methyladenosine,other RNA methylation modifications-such as m1A,m5C,m7G,uracil,and pseudouridine-are implicated in neurodegenerative diseases through their regulation of mitochondrial function,RNA metabolism,and neuronal stress responses.Additionally,N^(6)-methyladenosine exhibits cell type-specific functions:in microglia,it regulates inflammatory activation and phagocytic function;in astrocytes,it modulates metabolic homeostasis and glutamate-associated neurotoxicity;in neurons,it affects synaptic function and neurodegeneration-related gene expression;and in adult neural stem cells,it controls differentiation,neurogenesis,and cognitive plasticity.Recently,several small-molecule inhibitors targeting methyltransferase-like 3 or fat mass and obesity-associated protein have been developed to modulate N^(6)-methyladenosine modification,providing new opportunities for disease intervention,with the targeting of N⁶-methyladenosine-related pathways emerging as a promising therapeutic strategy.However,challenges persist in optimizing the specificity and delivery of these therapeutic approaches.展开更多
Cell cycle progression is regulated by interactions between cyclins and cyclin-dependent kinases (CDKs). p21(WAF1) is one of the CIP/KIP family which inhibits CDKs activity. Increased expression of p21(WAF1) may play ...Cell cycle progression is regulated by interactions between cyclins and cyclin-dependent kinases (CDKs). p21(WAF1) is one of the CIP/KIP family which inhibits CDKs activity. Increased expression of p21(WAF1) may play an important role in the growth arrest induced in transformed cells. Although the stability of the p21( WAF1) mRNA could be altered by different signals, cell differentiation and numerous influencing factors. However, recent studies suggest that two known mechanisms of epigenesis, i.e.gene inactivation by methylation in promoter region and changes to an inactive chromatin by histone deacetylation, seem to be the best candidate mechanisms for inactivation of p21( WAF1). To date, almost no coding region p21(WAF1) mutations have been found in tumor cells, despite extensive screening of hundreds of various tumors. Hypermethylation of the p21(WAF1) promoter region may represent an alternative mechanism by which the p21(WAF1/CIP1) gene can be inactivated. The reduction of cellular DNMT protein levels also induces a corresponding rapid increase in the cell cycle regulator p21(WAF1) protein demonstrating a regulatory link between DNMT and p21(WAF1) which is independent of methylation of DNA. Both histone hyperacetylation and hypoacetylation appear to be important in the carcinoma process, and induction of the p21(WAF1) gene by histone hyperacetylation may be a mechanism by which dietary fiber prevents carcinogenesis. Here, we review the influence of histone acetylation and DNA methylation on p21(WAF1) transcription, and affection of pathways or factors associated such as p 53, E2A, Sp1 as well as several histone deacetylation inhibitors.展开更多
BACKGROUND DNA methylation, acknowledged as a key modification in the field of epigenetics, regulates gene expression at the transcriptional level. Aberrant methylation in DNA regulatory regions could upregulate oncog...BACKGROUND DNA methylation, acknowledged as a key modification in the field of epigenetics, regulates gene expression at the transcriptional level. Aberrant methylation in DNA regulatory regions could upregulate oncogenes and downregulate tumor suppressor genes without changing the sequences.However, studies of methylation in the control of gene expression are still inadequate. In the present research, we performed bioinformatics analysis to clarify the function of methylation and supply candidate methylation-related biomarkers and drivers for colon cancer.AIM To identify and analyze methylation-regulated differentially expressed genes(MeDEGs) in colon cancer by bioinformatics analysis.METHODS We downloaded RNA expression profiles, Illumina Human Methylation 450 K BeadChip data, and clinical data of colon cancer from The Cancer Genome Atlas project. MeDEGs were identified by analyzing the gene expression and methylation levels using the edgeR and limma package in R software. Gene ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment analyses were performed in the DAVID database and KEGG Orthology-Based Annotation System 3.0, respectively. We then conducted Kaplan–Meier survival analysis to explore the relationship between methylation and expression and prognosis. Gene set enrichment analysis(GSEA) and investigation of protein-protein interactions(PPI) were performed to clarify the function of prognosis-related genes.RESULTS A total of 5 up-regulated and 81 down-regulated genes were identified asMeDEGs. GO and KEGG pathway analyses indicated that MeDEGs were enriched in multiple cancer-related terms. Furthermore, Kaplan–Meier survival analysis showed that the prognosis was negatively associated with the methylation status of glial cell-derived neurotrophic factor(GDNF) and reelin(RELN). In PPI networks, GDNF and RELN interact with neural cell adhesion molecule 1. Besides, GDNF can interact with GDNF family receptor alpha(GFRA1), GFRA2, GFRA3, and RET. RELN can interact with RAFAH1 B1,disabled homolog 1, very low-density lipoprotein receptor, lipoprotein receptorrelated protein 8, and NMDA 2 B. Based on GSEA, hypermethylation of GDNF and RELN were both significantly associated with pathways including "RNA degradation," "ribosome," "mismatch repair," "cell cycle" and "base excision repair."CONCLUSION Aberrant DNA methylation plays an important role in colon cancer progression.MeDEGs that are associated with the overall survival of patients may be potential targets in tumor diagnosis and treatment.展开更多
Salsolinol(1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline,Sal)is a catechol isoquinoline that causes neurotoxicity and shares structural similarity with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine,an environme...Salsolinol(1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline,Sal)is a catechol isoquinoline that causes neurotoxicity and shares structural similarity with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine,an environmental toxin that causes Parkinson's disease.However,the mechanism by which Sal mediates dopaminergic neuronal death remains unclear.In this study,we found that Sal significantly enhanced the global level of N~6-methyladenosine(m~6A)RNA methylation in PC12 cells,mainly by inducing the downregulation of the expression of m~6A demethylases fat mass and obesity-associated protein(FTO)and alk B homolog 5(ALKBH5).RNA sequencing analysis showed that Sal downregulated the Hippo signaling pathway.The m~6A reader YTH domain-containing family protein 2(YTHDF2)promoted the degradation of m~6A-containing Yes-associated protein 1(YAP1)mRNA,which is a downstream key effector in the Hippo signaling pathway.Additionally,downregulation of YAP1 promoted autophagy,indicating that the mutual regulation between YAP1 and autophagy can lead to neurotoxicity.These findings reveal the role of Sal on m~6A RNA methylation and suggest that Sal may act as an RNA methylation inducer mediating dopaminergic neuronal death through YAP1 and autophagy.Our results provide greater insights into the neurotoxic effects of catechol isoquinolines compared with other studies and may be a reference for assessing the involvement of RNA methylation in the pathogenesis of Parkinson's disease.展开更多
In vitro maturation(IvM)of human oocytes offers cost efficiency and minimal invasiveness,serving as a valuable supplementary tool in assisted reproduction for fertility preservation,ovarian hyperstimulation syndrome p...In vitro maturation(IvM)of human oocytes offers cost efficiency and minimal invasiveness,serving as a valuable supplementary tool in assisted reproduction for fertility preservation,ovarian hyperstimulation syndrome prevention,and other reproductive strategies.Despite its availability for three decades,the clinical use of IVM remains limited due to efficacy and safety concerns.This study examines the DNA methylation profile of IVM oocytes collected during laparoscopic/hysteroscopic surgeries compared to in vivo matured oocytes via reduced representation bisulfite sequencing.Results indicate IVM oocytes exhibit a higher global methylation level.Differentially methylated regions(DMRs)analysis reveals that the in vitro group displays more hypermethylated and fewer hypomethylated DMRs compared to the in vivo group.Additionally,the in vitro group exhibits a higher level of non-CpG methylation than the in vivo group.However,no significant correlation between methylation levels and transcriptional activity in these oocytes is found,especially for those specific imprinted genes or genes related to embryonic development.These findings shed light on the epigenetic landscape of IvM oocytes,contributing to the ongoing assessment of their clinical feasibility and safety in assisted reproduction.展开更多
INTRODUCTIONThe mechanism that DNA hypomethylation leads toactivation of oncogene and occurrence of malignantneoplasm is being increasingly recognized byresearchers. Normal DNA methylation playsimportant role in stabi...INTRODUCTIONThe mechanism that DNA hypomethylation leads toactivation of oncogene and occurrence of malignantneoplasm is being increasingly recognized byresearchers. Normal DNA methylation playsimportant role in stabilizing the phenotype of cell.DNA methylation status reduction and/or patternalteration are related to activation and abnormallyhigh expression of some oncogenes and cellularmalignancy[1-6]. c-fms oncogene encodes for colonystimulating factor 1 receptor (CSF-1R)[7], c-fms/CSF-1R was highly expressed in hepatocellularcarcinoma (HCC) tissue, but the mechanismremained obscure[8,9].展开更多
DNA methylation is an important promising biomarker for cancer diagnosis and monitoring.Therefore,the assessment of DNA methylation levels is helpful for the prognosis and diagnosis of cancer.However,it is still a hug...DNA methylation is an important promising biomarker for cancer diagnosis and monitoring.Therefore,the assessment of DNA methylation levels is helpful for the prognosis and diagnosis of cancer.However,it is still a huge challenge to sensitively and accurately quantify the levels of DNA methylation in clinical sample.In this work,we proposed a protospacer adjacent motif(PAM)-free mediated CRISPR-Cas12a ultra-sensitive and quantitative DNA methylation detection method.Through recognizing the ds DNA with toehold region,CRISPR-Cas12a not only got rid of the limitation of PAM,but also improved its distinction ability for single Cp G site methylation,nearly 5-fold that of conventional PAM-containing ds DNA.We further introduced assist-strand and design an artificial mismatch to greatly improve the ability to distinguish single Cp G methylation site.Our results showed that the discrimination factor was >200.Then,we constructed toe-ds DNA by using “heating and freezing”,which made our method universally applicable and feasible.In addition,we greatly simplified the difficulty of primer design.Our method detected four highly methylated genes acyl carrier protein(ACP),CLV3/ESR-related(CLE),Disabled(DAB) and Homeobox(HOX) with a detection limit of 0.01 % and excellent linearity in DNA methylation standards.Then,we verified the clinical utility of this method in 29 hepatocellular carcinomas,11 ovarian cancers and4 health people.In conclusion,we have successfully constructed a PAM-free CRISPR-Cas12a DNA methylation quantification method,which achieves high congruence in sensitivity,specificity and universality,fully demonstrating its significant clinical application value.展开更多
Fig(Ficus carica L.)with purple-red peel cultivars are popular among consumers and exhibit better storability.While DNA methylation influences fruit ripening and color development,its specific role in fig fruit remain...Fig(Ficus carica L.)with purple-red peel cultivars are popular among consumers and exhibit better storability.While DNA methylation influences fruit ripening and color development,its specific role in fig fruit remains unclear.This study explores the impact of DNA methylation on the fig peel coloration.Enzymatic colorimetric detection revealed that the level of‘Purple Peel’fig DNA methylation decreases with fig fruit ripening and coloring.Treatment of young fruit with the DNA-methylation inhibitor azacytidine induced peel coloration,suggesting that a decrease in DNA-methylation level promotes fig peel coloration.Seven members of DNA methyltransferases and three members of DNA demethylases were identified from a high-level fig genome,highlighting FcMET1 and FcDRM2 as stable proteins,ensuring functional expression.Reference to the Arabidopsis protein interaction network map predicted that FcMET1 is in a central position,suggesting a crucial regulatory role in multiple biological processes.Correlation analysis revealed a positive correlation between FcMET1 expression during peel development and the level of total DNA methylation.Weighted gene co-expression network analysis identified co-expression of FcMET1 with the color-related transcription factors MYB,bHLH and WD40,as well as with eight structural genes in the flavonoid-biosynthesis pathway.The expression of FcUFGT3 was negatively correlated with that of FcMET1.McrBC-PCR and Bisulfite Sequencing detection showed that a low methylation level of the FcUFGT3 promoter corresponds with its high expression in colored fig.This investigation of the mechanism of DNA methylation provides a theoretical basis for understanding the role of DNA-methylation modifications in fig ripening and coloring.展开更多
Objective:To discuss the effect of insulin and metformin on amethylation and glycolipid metabolism of peroxisome proliferator-activated receptor γ coactivator-1A(PPARGC1A) of rat offspring with gestational diabetes m...Objective:To discuss the effect of insulin and metformin on amethylation and glycolipid metabolism of peroxisome proliferator-activated receptor γ coactivator-1A(PPARGC1A) of rat offspring with gestational diabetes mellitus(GDM).Methods:A total of 45 pregnant rats received the intraperitoneal injection of streptozotocin to establish the pregnant rat model of GDM.A total of 21 pregnant rats with GDM were randomly divided into three groups,with 7ruts in each group,namely the insulin group,metformin group and control group.Rats in the insulin group received the abdominal subcutaneous injection of 1 mL/kg recombinant insulin glargine at 18:00 every day.Rats in the metformin group received the intragastric infusion of metformin hydrochloride at 18:00 every day,with the first dose of 300 mg/kg.The doses of two groups were adjusted every 3 d to maintain the blood glucose level at 2.65-7.62 mmol/L.Rats in the control group received the intragastric infusion of 1 mL normal saline at 18:00 every day.After the natural delivery of pregnant rats.10 offspring rats were randomly selected from each group.At birth,4 wk and 8 wk after the birth of offspring rats,the weight of offspring rats was measured.The blood glucose level of offspring rats was measured at 4wk and 8 wk,while the level of serum insulin,triglyceride and leptin was measured at 8 wk.Results:The weight of offspring rats at birth in the insulin group and metformin group was significantly lower than the one in the control group(P<0.05),and there was no significant difference at 4 wk and 8 wk among three groups(P>0.05).The fasting blood glucose and random blood glucose in the insulin group and metformin group at 4 wk and 8 wk were all significantly lower than ones in the control group(P<0.05);there was no significant difference between the insulin group and metformin group(P>0.05).The expression of PPARGC1 A mRNA in the insulin group and metformin group was significantly higher and the methylation level of PPARGC1 A was significantly lower than the one in the control group(P<0.05),but there was no significant difference between the insulin group and metformin group(P>0.05).Insulin and leptin at 8 wk in the insulin group and metformin group were significantly higher,while triglyceride was significantly lower than the one in the control group(P<0.05);triglyceride level of rats in the insulin group was significantly higher than the one in the metformin group(P<0.05).There was no significant difference in insulin and leptin level of offspring rats between the insulin group and metformin group(P>0.05).Conclusions:GDM can induce the methylation of PPARGC1 A of offspring rats to reduce the expression of PPARGC1 A mRNA and then cause the disorder of glycolipid metabolism when the offspring rats grow up;the insulin or metformin in the treatment of pregnant rats with GDM can reduce the methylation level of PPARGC1 A and thus improve the abnormal glycolipid metabolism of offspring rats.展开更多
AIM:To explore the possibility that PDX-1 gene is reactivated as a consequence of molecular events that occur during liver regeneration. METHODS:Rat hepatocytes were maintained in DMEM- F12,10%fetal bovine serum(FBS),...AIM:To explore the possibility that PDX-1 gene is reactivated as a consequence of molecular events that occur during liver regeneration. METHODS:Rat hepatocytes were maintained in DMEM- F12,10%fetal bovine serum(FBS),penicillin/streptomycin and geneticin when applicable.Rat insulinoma RIN 1046-38 cells were maintained in M-199-10%FBS and penicillin/streptomycin.The final concentration of glucose was 11.1 mmol/L.During regeneration,lateral and medial liver lobes of adult male Wistar rats were surgically removed,with up 70%loss of liver mass.In methylation experiments,5-aza-deoxycytidine(5-aza-dC)was used.Primer3 software was used for poly- merase chain reaction(PCR).Quantitative real time PCR (qRT-PCR)was performed using SYBR Green technol- ogy;primers were designed by Beacon Designer 6 software.Western blotting and SDS-PAGE were performed according to standard procedures.Antibodies were purchased from commercial suppliers.RESULTS:We explored the possibility that liver regeneration could trigger PDX-1 expression,and hence insulin production.Twenty-four hours after surgical liver removal,regeneration was active as demonstrated by the increased proliferating cell nuclear antigen;however, all the other checked genes(involved in insulin gene expression):PC-1,Ngn3,NeuroD1,Btc,PDX-1 and Ins-1, were not related to the molecular events caused by this process.The only marker detected in regenerating liver was E47:a transcription factor of the the basic helixloop-helix family known to be expressed ubiquitously in mammalian cells.In the rat pancreas,almost all of the tested genes were expressed as shown by RT-PCR, except for Ngn3,which was silenced 2 d after birth. Therefore,the molecular events in liver regeneration are not sufficient to promote PDX-1 expression.DNA methylation is a known mechanism to achieve stable re- pression of gene expression in mammals:Hxk 2 gene is silenced through this mechanism in normal hepatocytes. The administration of 5-aza-dC to cultured cells is in fact able to upregulate Hxk 2 mRNA.We investigated whether PDX-1 silencing in liver cells could be exerted through methylation of CpG islands in both the promoter and the gene coding regions.The results show that the drug increased the expression level of the Hxk 2 control gene but failed to rescue the expression of PDX-1,thus DNA demethylation is not sufficient to override repression of the PDX-1 gene.展开更多
AIM: To evaluate tumor necrosis factor-α converting enzyme(TACE) methylation status in patients with chronic hepatitis B(CHB).METHODS: Eighty patients with hepatitis B e antigen(HBe Ag)-positive CHB, 80 with HBe Ag-n...AIM: To evaluate tumor necrosis factor-α converting enzyme(TACE) methylation status in patients with chronic hepatitis B(CHB).METHODS: Eighty patients with hepatitis B e antigen(HBe Ag)-positive CHB, 80 with HBe Ag-negative CHB, and 40 healthy controls(HCs) were randomly enrolled in this study. Genomic DNA was extracted from peripheral blood mononuclear cells and methylation status of TACE promoter was determined by methylation-specific polymerase chain reaction. The clinical and laboratory parameters were collected.RESULTS: One hundred and thirty of 160 patients with CHB(81.25%) and 38 of 40 HCs(95%) displayed TACE promoter methylation. The difference was significant(χ2 = 4.501, P < 0.05). TACE promoter methylation frequency in HBe Ag-positive CHB(58/80, 72.5%) was significantly lower than that in HBe Ag-negative CHB(72/80, 90%; χ2 = 8.041, P < 0.01) and HCs(χ2 = 8.438, P < 0.01). However, no significant difference was observed in the methylation frequency between HBe Agnegative CHB and HCs(χ2 = 0.873, P > 0.05). In the HBe Ag-positive group, TACE methylation frequency was significantly negatively correlated with HBe Ag(r =-0.602, P < 0.01), alanine aminotransferase(r =-0.461, P < 0.01) and aspartate aminotransferase(r =-0.329, P < 0.01). CONCLUSION: Patients with HBe Ag-positive CHB have aberrant demethylation of the TACE promoter, which may potentially serve as a biomarker for HBe Ag seroconversion.展开更多
AIM:To evaluate the utility of measuring epigenetic alterations in pancreatic and biliary fluids in determining molecular markers for pancreatobiliary cancers.METHODS:DNA was extracted from undiluted pancreatic and bi...AIM:To evaluate the utility of measuring epigenetic alterations in pancreatic and biliary fluids in determining molecular markers for pancreatobiliary cancers.METHODS:DNA was extracted from undiluted pancreatic and biliary fluids.As a surrogate for a genomewide hypomethylation assay,levels of long interspersed nuclear element-1(LINE-1) methylation were analyzed using bisulfite pyrosequencing.CpG island hypermethylation of 10 tumor-associated genes,aryl-hydrocarbon receptor repressor,adenomatous polyposis coli,calcium channel,voltage dependent,T type α1G subunit,insulin-like growth factor 2,O-6-methyl-guanine-DNA methyltransferase,neurogenin 1,CDKN2A,runt-related transcription factor 3(RUNX3),secreted frizzled-related protein 1,and ubiquitin carboxyl-terminal esterase L1(UCHL1),was analyzed using MethyLight.To examine the role of CpG methylation and histone deacetylation in the silencing of UCHL1,human gallbladder carcinoma cell lines and pancreatic carcinoma cell lines were treated with 2 or 5 μmol/L 5-AZA-dC for 72 h or 100 nmol/L Trichostatin A for 24 h.After the treatment,UCHL1 expression was analyzed by real-time reverse transcription-polymerase chain reaction.RESULTS:Pancreatobiliary cancers exhibited significantly lower LINE-1 methylation levels in pancreatic and biliary fluids than did noncancerous pancreatobiliary disease(58.7% ± 4.3% vs 61.7% ± 2.2%,P = 0.027;53.8% ± 6.6% vs 57.5% ± 1.7%,P = 0.007);however,LINE-1 hypomethylation was more evident in pancreatic cancer tissues than in pancreatic fluids(45.4% ± 5.5% vs 58.7% ± 4.3%,P < 0.001).CpG island hypermethylation of tumor-associated genes was detected at various frequencies,but it was not correlated with LINE-1 hypomethylation.Hypermethylation of the UCHL1 gene was cancer-specific and most frequently detected in pancreatic(67%) or biliary(70%) fluids from patients with pancreatobiliary cancer.As a single marker,hypermethylation of the UCHL1 gene in pancreatic and biliary fluids was most useful for the detection of pancreatic and pancreatobiliary cancers,respectively(100% specificity).Hypermethylation of the UCHL1 and RUNX3 genes in pancreatic and biliary fluids was the most useful combined marker for pancreatic(87% sensitivity and 100% specificity) and pancreatobiliary(97% sensitivity and 100% specificity) cancers.Treatment with a demethylating agent,5-AZA-2'-deoxycytidine,restored UCHL1 expression in pancreatobiliary cancer cell lines.CONCLUSION:Our results suggest that hypermethylation of UCHL1 and RUNX3 in pancreatobiliary fluid might be useful for the diagnosis of pancreatobiliary cancers.展开更多
In this study,the methylation-sensitive amplification polymorphism(MSAP)was used to compare the genomic DNA methylation level of muscle,gill and hepatopancreas of Portunus trituberculatus subjected to salinity 12 for ...In this study,the methylation-sensitive amplification polymorphism(MSAP)was used to compare the genomic DNA methylation level of muscle,gill and hepatopancreas of Portunus trituberculatus subjected to salinity 12 for 30 days to illustrate the epigenetic mechanism of osmoregulation.Thirty primers were used to analyze the difference of methylation level of different tissues.The results showed that the baseline methylation level of muscle,hepatopancreas and gill was 47.31%,22.94%and 17.69%,respectively.After exposed to low salinity stress,the methylation epiloci changed in the three tissues.Both demethylation and methylation processes occurred under low salinity stress.The methylation ratio decreased in muscle and gill but increased in hepatopancreas.These results indicated that DNA methylation is tissue-specific when P.trituberculatus responds to low salinity.展开更多
Epigenetic changes of DNA, including methylation, have long been recognized as key indicators of various diseases, including aging, cancer, and neurological disorders. Biomarker discoveries based on distinct methylati...Epigenetic changes of DNA, including methylation, have long been recognized as key indicators of various diseases, including aging, cancer, and neurological disorders. Biomarker discoveries based on distinct methylation patterns for both hypermethylation and hypomethylation lead the way in discovery of novel diagnosis and treatment targets. Many different approaches are present to detect the level of methylation in whole genome (whole genome bisulfite sequencing, microarray) as well as at specific loci (methylation specific PCR). Cell-free DNA (cf-DNA) found in body fluids like blood provides information about DNA methylation and serves as a less invasive approach for genetic screening. Cell-free DNA and methylation screening technologies, when combined, have the potential to transform the way we approach genetic screening and personalized therapy. These technologies can help enhance disease diagnostic accuracy and inform the development of targeted therapeutics by providing a non-invasive way for acquiring genomic information and identifying disease-associated methylation patterns. We highlight the clinical benefits of using cell-free DNA (cf-DNA) liquid biopsy analysis and available methylation screening technologies that have been crucial in identifying biomarkers for disease from patients using a non-invasive way. Powering such biomarker discoveries are various methods of cf-DNA methylation analysis such as Bisulfite Sequencing and most recently, Methylation-Specific Restriction Enzyme (MSRE-seq) Analysis, paving the way for novel epigenetic biomarker discoveries for more robust diagnosis such as early disease detection, prognosis, monitoring of disease progression and treatment response as well as discovery of novel drug targets.展开更多
The tumor selectivity of alkylating agents that produce guanine O6-chloroethyl (laromustine and carmustine) and O6-methyl (temozolomide) lesions depends upon O6-methylguanine-DNA methyltransferase (MGMT) activity bein...The tumor selectivity of alkylating agents that produce guanine O6-chloroethyl (laromustine and carmustine) and O6-methyl (temozolomide) lesions depends upon O6-methylguanine-DNA methyltransferase (MGMT) activity being lower in tumor than in host tissue. Despite the established role of MGMT as a tumor resistance factor, consensus on how to assess MGMT expression in clinical samples is unsettled. The aim of this study is to examine the relationship between the values derived from distinctive MGMT measurements in 13, 12, 6 and 2 pairs of human tumors and matched normal adjacent tissue from the colon, kidney, lung and liver, respectively, and in human cell lines. The MGMT measurements included 1) alkyl-transfer assays using [benzene-3H]O6-benzylguanine as a substrate to assess functional MGMT activity, 2) methylation-specific PCR (MSP) to probe MGMT gene promoter CpG methylations as a measure of gene silencing, and 3) western immunoblots to analyze the MGMT protein. In human cell lines, a strict negative correlation existed between MGMT activity and the extent of promoter methylation. In tissue specimens, by contrast, the correlation between these two variables was low. Moreover, alkyl-transfer assays identified 3 pairs of tumors and normal tissue with tumor-selective reduction in MGMT activity in the absence of promoter methylation. Cell line MGMT migrated as a single band in western analyses, whereas tissue MGMT was heterogeneous around its molecular size and at much higher molecular masses, indicative of multi-layered post-translational modifications. Malignancy is occasionally associated with a mobility shift in MGMT. Contrary to the prevalent expectation that MGMT expression is governed at the level of gene silencing, these data suggest that other mechanisms that can lead to tumorselective reduction in MGMT activity exist in human tissue.展开更多
Following the publication of Xu et al.(2022),an error was identified in Figure 1D.Specifically,the top left panel was inadvertently duplicated during figure preparation.To ensure the accuracy and integrity of our publ...Following the publication of Xu et al.(2022),an error was identified in Figure 1D.Specifically,the top left panel was inadvertently duplicated during figure preparation.To ensure the accuracy and integrity of our published work,we request the publication of a corrigendum with the corrected image.We apologize for this oversight and any confusion it may have caused.The amended figure is provided in the updated Supplementary Materials.展开更多
BACKGROUND Epigenetic regulation of leptin(LEP)plays a critical role in metabolic disorders,yet its promoter methylation patterns in lean diabetic populations remain poorly characterized.Emerging evidence suggests DNA...BACKGROUND Epigenetic regulation of leptin(LEP)plays a critical role in metabolic disorders,yet its promoter methylation patterns in lean diabetic populations remain poorly characterized.Emerging evidence suggests DNA methylation may precede clinical hyperglycemia,offering potential for early risk stratification.While obesity-associated LEP methylation is well-studied,lean Asian populations who exhibit high diabetes prevalence despite lower adiposity,represent an underexplored cohort.This study hypothesizes that LEP promoter methylation in peripheral leukocytes decreases progressively from normoglycemia to prediabetes and type 2 diabetes mellitus(T2DM),correlating inversely with serum LEP levels in lean Chinese adults[body mass index(BMI)<24 kg/m^(2)].AIM To investigate LEP promoter methylation status and its association with serum LEP levels across glycemic states in lean Chinese adults.METHODS We enrolled 392 participants including 120 normoglycemic controls,94 prediabetes[44 impaired fasting glucose(IFG)/50 impaired glucose tolerance(IGT)],178 T2DM aged 40-60 years with BMI<24 kg/m^(2).Genomic DNA from peripheral leukocytes underwent bisulfite conversion followed by methylation-specific PCR to assess CpG methylation in the LEP promoter.Serum LEP was quantified via enzyme-linked immunosorbent assay,with other parameters measured through standard assays.Statistical analyses included analysis of variance,χ²tests,and Pearson correlation(Bonferroni-corrected P value).RESULTS Methylation frequencies declined progressively:59.2%(controls)reduced to 43.6%(prediabetes;IFG:38.6%,IGT:48%)reduced to 31.5%(T2DM)(all P<0.05 vs controls;T2DM vs IGT:P=0.030).Serum LEP levels increased significantly in T2DM(16.94±4.19μg/L)vs controls(11.33±3.10μg/L;P=0.002),with intermediate values in prediabetes(IFG:13.79±3.32μg/L;IGT:12.62±4.81μg/L).A near-perfect inverse correlation between methylation and LEP levels was observed(r=-0.95,95%CI:-0.97 to-0.92,P<0.001),persisting after adjusting for age and BMI(β=-0.91,P<0.001).CONCLUSION LEP promoter hypomethylation parallels worsening glycemic status in lean Chinese adults,suggesting its potential as a blood-based epigenetic biomarker for diabetes progression,pending validation in longitudinal cohorts.展开更多
In the mammalian genome,most CpGs are methylated.However,CpGs within the CpG islands(CGIs)are largely unmethylated,which are important for gene expression regulation.The mechanism underlying the low methylation levels...In the mammalian genome,most CpGs are methylated.However,CpGs within the CpG islands(CGIs)are largely unmethylated,which are important for gene expression regulation.The mechanism underlying the low methylation levels at CGIs remains largely elusive.KDM2 proteins(KDM2A and KDM2B)are H3K36me2 demethylases known to bind specifically at CGIs.Here,we report that depletion of each or both KDM2 proteins,or mutation of all their JmjC domains that harbor the H3K36me2 demethylation activity,leads to an increase in DNA methylation at selective CGIs.The Kdm2a/2b double knockout shows a stronger increase in DNA methylation compared with the single mutant of Kdm2a or Kdm2b,indicating that KDM2A and KDM2B redundantly regulate DNA methylation at CGIs.In addition,the increase of CGI DNA methylation upon mutations of KDM2 proteins is associated with the chromatin environment.Our findings reveal that KDM2A and KDM2B function redundantly in regulating DNA methylation at a subset of CGIs in an H3K36me2 demethylation-dependent manner.展开更多
基金supported by the National Natural Science Foundation of China,No.81671469,81171072(to ZWY)the National Basic Research Program of China(973 Program),No.2013CB945402(to ZWY)the Program for Liaoning Innovative Research Team in University of China,No.LT2013016(to ZWY)
文摘O6-methylguanine DNA methyltransferase(MGMT), a DNA repair enzyme, has been reported in some congenital malformations, but it is less frequently reported in neural tube defects. This study investigated MGMT mRNA expression and methylation levels in the early embryo and in different embryonic stages, as well as the relationship between MGMT and neural tube defects. Spina bifida aperta was induced in rats by a single intragastric administration of all-trans retinoic acid on embryonic day(E) 10, whereas normal control rats received the same amount of olive oil on the same embryonic day. DNA damage was assessed by detecting γ-H2 A.X in spina bifida aperta rats. Real time-polymerase chain reaction was used to examine mRNA expression of MGMT in normal control and spina bifida aperta rats. In normal controls, the MGMT mRNA expression decreased with increasing embryonic days, and was remarkably reduced from E11 to E14, reaching a minimum at E18. In the spina bifida aperta model, γ-H2 A.X protein expression was increased, and mRNA expression of MGMT was markedly decreased on E14, E16, and E18. Bisulfite sequencing polymerase chain reaction for MGMT promoter methylation demonstrated that almost all CpG sites in the MGMT promoter remained unmethylated in both spina bifida aperta rats and normal controls, and there was no significant difference in methylation level between the two groups on either E14 or E18. Our results show that DNA damage occurs in spina bifida aperta rats. The mRNA expression of MGMT is downregulated, and this downregulation is independent of promoter DNA methylation.
基金supported by the National Nature Science Foundation of China(General Program),Nos.82271237,82071218(both to JC),and 82230042(to ZY)the Foundation of Key Laboratory of Neurology,Hebei Medical University,Ministry of Education,China,No.2023001(to JC).
文摘N^(6)-methyladenosine RNA methylation,an essential post-transcriptional modification,dynamically regulates RNA metabolism and plays a crucial role in neuronal function.Growing evidence suggests that dysregulated N^(6)-methyladenosine modification contributes to the pathogenesis of neurodegenerative diseases,including Alzheimer’s disease,Parkinson’s disease,multiple sclerosis,and amyotrophic lateral sclerosis.However,the precise mechanisms by which N^(6)-methyladenosine modification influences these conditions remain unclear.This review summarizes the role of m6A modification and its associated regulators in neurodegeneration,focusing on their involvement in key pathological processes.In Alzheimer’s disease,m6A modification contributes to synaptic dysfunction,mitochondrial damage,and neuronal apoptosis.Evidence from APP/PS1,5xFAD,tau transgenic,and Drosophila models demonstrates that regulators such as methyltransferase-like 3 and fat mass and obesity-associated protein influence Alzheimer’s disease progression through neuroinflammation,circular RNAs dysregulation,and autophagy-related mechanisms.In Parkinson’s disease,altered N^(6)-methyladenosine regulator expression affects dopaminergic neuron survival and stress responses by modulating mRNA stability and autophagy-related lncRNAs.In multiple sclerosis and amyotrophic lateral sclerosis,N^(6)-methyladenosine affects immune activation,myelin repair,and the regulation of disease-associated genes such as TDP-43.Beyond N^(6)-methyladenosine,other RNA methylation modifications-such as m1A,m5C,m7G,uracil,and pseudouridine-are implicated in neurodegenerative diseases through their regulation of mitochondrial function,RNA metabolism,and neuronal stress responses.Additionally,N^(6)-methyladenosine exhibits cell type-specific functions:in microglia,it regulates inflammatory activation and phagocytic function;in astrocytes,it modulates metabolic homeostasis and glutamate-associated neurotoxicity;in neurons,it affects synaptic function and neurodegeneration-related gene expression;and in adult neural stem cells,it controls differentiation,neurogenesis,and cognitive plasticity.Recently,several small-molecule inhibitors targeting methyltransferase-like 3 or fat mass and obesity-associated protein have been developed to modulate N^(6)-methyladenosine modification,providing new opportunities for disease intervention,with the targeting of N⁶-methyladenosine-related pathways emerging as a promising therapeutic strategy.However,challenges persist in optimizing the specificity and delivery of these therapeutic approaches.
文摘Cell cycle progression is regulated by interactions between cyclins and cyclin-dependent kinases (CDKs). p21(WAF1) is one of the CIP/KIP family which inhibits CDKs activity. Increased expression of p21(WAF1) may play an important role in the growth arrest induced in transformed cells. Although the stability of the p21( WAF1) mRNA could be altered by different signals, cell differentiation and numerous influencing factors. However, recent studies suggest that two known mechanisms of epigenesis, i.e.gene inactivation by methylation in promoter region and changes to an inactive chromatin by histone deacetylation, seem to be the best candidate mechanisms for inactivation of p21( WAF1). To date, almost no coding region p21(WAF1) mutations have been found in tumor cells, despite extensive screening of hundreds of various tumors. Hypermethylation of the p21(WAF1) promoter region may represent an alternative mechanism by which the p21(WAF1/CIP1) gene can be inactivated. The reduction of cellular DNMT protein levels also induces a corresponding rapid increase in the cell cycle regulator p21(WAF1) protein demonstrating a regulatory link between DNMT and p21(WAF1) which is independent of methylation of DNA. Both histone hyperacetylation and hypoacetylation appear to be important in the carcinoma process, and induction of the p21(WAF1) gene by histone hyperacetylation may be a mechanism by which dietary fiber prevents carcinogenesis. Here, we review the influence of histone acetylation and DNA methylation on p21(WAF1) transcription, and affection of pathways or factors associated such as p 53, E2A, Sp1 as well as several histone deacetylation inhibitors.
文摘BACKGROUND DNA methylation, acknowledged as a key modification in the field of epigenetics, regulates gene expression at the transcriptional level. Aberrant methylation in DNA regulatory regions could upregulate oncogenes and downregulate tumor suppressor genes without changing the sequences.However, studies of methylation in the control of gene expression are still inadequate. In the present research, we performed bioinformatics analysis to clarify the function of methylation and supply candidate methylation-related biomarkers and drivers for colon cancer.AIM To identify and analyze methylation-regulated differentially expressed genes(MeDEGs) in colon cancer by bioinformatics analysis.METHODS We downloaded RNA expression profiles, Illumina Human Methylation 450 K BeadChip data, and clinical data of colon cancer from The Cancer Genome Atlas project. MeDEGs were identified by analyzing the gene expression and methylation levels using the edgeR and limma package in R software. Gene ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment analyses were performed in the DAVID database and KEGG Orthology-Based Annotation System 3.0, respectively. We then conducted Kaplan–Meier survival analysis to explore the relationship between methylation and expression and prognosis. Gene set enrichment analysis(GSEA) and investigation of protein-protein interactions(PPI) were performed to clarify the function of prognosis-related genes.RESULTS A total of 5 up-regulated and 81 down-regulated genes were identified asMeDEGs. GO and KEGG pathway analyses indicated that MeDEGs were enriched in multiple cancer-related terms. Furthermore, Kaplan–Meier survival analysis showed that the prognosis was negatively associated with the methylation status of glial cell-derived neurotrophic factor(GDNF) and reelin(RELN). In PPI networks, GDNF and RELN interact with neural cell adhesion molecule 1. Besides, GDNF can interact with GDNF family receptor alpha(GFRA1), GFRA2, GFRA3, and RET. RELN can interact with RAFAH1 B1,disabled homolog 1, very low-density lipoprotein receptor, lipoprotein receptorrelated protein 8, and NMDA 2 B. Based on GSEA, hypermethylation of GDNF and RELN were both significantly associated with pathways including "RNA degradation," "ribosome," "mismatch repair," "cell cycle" and "base excision repair."CONCLUSION Aberrant DNA methylation plays an important role in colon cancer progression.MeDEGs that are associated with the overall survival of patients may be potential targets in tumor diagnosis and treatment.
基金supported by the National Natural Science Foundation of China,Nos.82271283(to XC),91854115(to JW),31970044(to JW)the Natural Science Foundation of Beijing,No.7202001(to XC)the Scientific Research Project of Beijing Educational Committee,No.KM202010005022(to XC)。
文摘Salsolinol(1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline,Sal)is a catechol isoquinoline that causes neurotoxicity and shares structural similarity with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine,an environmental toxin that causes Parkinson's disease.However,the mechanism by which Sal mediates dopaminergic neuronal death remains unclear.In this study,we found that Sal significantly enhanced the global level of N~6-methyladenosine(m~6A)RNA methylation in PC12 cells,mainly by inducing the downregulation of the expression of m~6A demethylases fat mass and obesity-associated protein(FTO)and alk B homolog 5(ALKBH5).RNA sequencing analysis showed that Sal downregulated the Hippo signaling pathway.The m~6A reader YTH domain-containing family protein 2(YTHDF2)promoted the degradation of m~6A-containing Yes-associated protein 1(YAP1)mRNA,which is a downstream key effector in the Hippo signaling pathway.Additionally,downregulation of YAP1 promoted autophagy,indicating that the mutual regulation between YAP1 and autophagy can lead to neurotoxicity.These findings reveal the role of Sal on m~6A RNA methylation and suggest that Sal may act as an RNA methylation inducer mediating dopaminergic neuronal death through YAP1 and autophagy.Our results provide greater insights into the neurotoxic effects of catechol isoquinolines compared with other studies and may be a reference for assessing the involvement of RNA methylation in the pathogenesis of Parkinson's disease.
基金supported by funding from the National Natural Science Foundation of China(81971349 and 81300456).
文摘In vitro maturation(IvM)of human oocytes offers cost efficiency and minimal invasiveness,serving as a valuable supplementary tool in assisted reproduction for fertility preservation,ovarian hyperstimulation syndrome prevention,and other reproductive strategies.Despite its availability for three decades,the clinical use of IVM remains limited due to efficacy and safety concerns.This study examines the DNA methylation profile of IVM oocytes collected during laparoscopic/hysteroscopic surgeries compared to in vivo matured oocytes via reduced representation bisulfite sequencing.Results indicate IVM oocytes exhibit a higher global methylation level.Differentially methylated regions(DMRs)analysis reveals that the in vitro group displays more hypermethylated and fewer hypomethylated DMRs compared to the in vivo group.Additionally,the in vitro group exhibits a higher level of non-CpG methylation than the in vivo group.However,no significant correlation between methylation levels and transcriptional activity in these oocytes is found,especially for those specific imprinted genes or genes related to embryonic development.These findings shed light on the epigenetic landscape of IvM oocytes,contributing to the ongoing assessment of their clinical feasibility and safety in assisted reproduction.
基金Supported by the Natural Science Foundation of Guangdong Province, No 990422
文摘INTRODUCTIONThe mechanism that DNA hypomethylation leads toactivation of oncogene and occurrence of malignantneoplasm is being increasingly recognized byresearchers. Normal DNA methylation playsimportant role in stabilizing the phenotype of cell.DNA methylation status reduction and/or patternalteration are related to activation and abnormallyhigh expression of some oncogenes and cellularmalignancy[1-6]. c-fms oncogene encodes for colonystimulating factor 1 receptor (CSF-1R)[7], c-fms/CSF-1R was highly expressed in hepatocellularcarcinoma (HCC) tissue, but the mechanismremained obscure[8,9].
基金financially supported by the Natural Science Foundation of Wuhan City (Chenguang Project) (No.2024040801020331)the Natural Science Foundation of Hubei Province of China (No.2023AFB402)+1 种基金the National Key Research and Development Program of China (No.2023YFE0210200)Interdisciplinary Research Program of HUST。
文摘DNA methylation is an important promising biomarker for cancer diagnosis and monitoring.Therefore,the assessment of DNA methylation levels is helpful for the prognosis and diagnosis of cancer.However,it is still a huge challenge to sensitively and accurately quantify the levels of DNA methylation in clinical sample.In this work,we proposed a protospacer adjacent motif(PAM)-free mediated CRISPR-Cas12a ultra-sensitive and quantitative DNA methylation detection method.Through recognizing the ds DNA with toehold region,CRISPR-Cas12a not only got rid of the limitation of PAM,but also improved its distinction ability for single Cp G site methylation,nearly 5-fold that of conventional PAM-containing ds DNA.We further introduced assist-strand and design an artificial mismatch to greatly improve the ability to distinguish single Cp G methylation site.Our results showed that the discrimination factor was >200.Then,we constructed toe-ds DNA by using “heating and freezing”,which made our method universally applicable and feasible.In addition,we greatly simplified the difficulty of primer design.Our method detected four highly methylated genes acyl carrier protein(ACP),CLV3/ESR-related(CLE),Disabled(DAB) and Homeobox(HOX) with a detection limit of 0.01 % and excellent linearity in DNA methylation standards.Then,we verified the clinical utility of this method in 29 hepatocellular carcinomas,11 ovarian cancers and4 health people.In conclusion,we have successfully constructed a PAM-free CRISPR-Cas12a DNA methylation quantification method,which achieves high congruence in sensitivity,specificity and universality,fully demonstrating its significant clinical application value.
基金supported by 111 Project(Grant No.B17043)China Postdoctoral Science Foundation(Grant No.2022M723425).
文摘Fig(Ficus carica L.)with purple-red peel cultivars are popular among consumers and exhibit better storability.While DNA methylation influences fruit ripening and color development,its specific role in fig fruit remains unclear.This study explores the impact of DNA methylation on the fig peel coloration.Enzymatic colorimetric detection revealed that the level of‘Purple Peel’fig DNA methylation decreases with fig fruit ripening and coloring.Treatment of young fruit with the DNA-methylation inhibitor azacytidine induced peel coloration,suggesting that a decrease in DNA-methylation level promotes fig peel coloration.Seven members of DNA methyltransferases and three members of DNA demethylases were identified from a high-level fig genome,highlighting FcMET1 and FcDRM2 as stable proteins,ensuring functional expression.Reference to the Arabidopsis protein interaction network map predicted that FcMET1 is in a central position,suggesting a crucial regulatory role in multiple biological processes.Correlation analysis revealed a positive correlation between FcMET1 expression during peel development and the level of total DNA methylation.Weighted gene co-expression network analysis identified co-expression of FcMET1 with the color-related transcription factors MYB,bHLH and WD40,as well as with eight structural genes in the flavonoid-biosynthesis pathway.The expression of FcUFGT3 was negatively correlated with that of FcMET1.McrBC-PCR and Bisulfite Sequencing detection showed that a low methylation level of the FcUFGT3 promoter corresponds with its high expression in colored fig.This investigation of the mechanism of DNA methylation provides a theoretical basis for understanding the role of DNA-methylation modifications in fig ripening and coloring.
基金supported by Shandong Natural Science Fund(Y2008c170)
文摘Objective:To discuss the effect of insulin and metformin on amethylation and glycolipid metabolism of peroxisome proliferator-activated receptor γ coactivator-1A(PPARGC1A) of rat offspring with gestational diabetes mellitus(GDM).Methods:A total of 45 pregnant rats received the intraperitoneal injection of streptozotocin to establish the pregnant rat model of GDM.A total of 21 pregnant rats with GDM were randomly divided into three groups,with 7ruts in each group,namely the insulin group,metformin group and control group.Rats in the insulin group received the abdominal subcutaneous injection of 1 mL/kg recombinant insulin glargine at 18:00 every day.Rats in the metformin group received the intragastric infusion of metformin hydrochloride at 18:00 every day,with the first dose of 300 mg/kg.The doses of two groups were adjusted every 3 d to maintain the blood glucose level at 2.65-7.62 mmol/L.Rats in the control group received the intragastric infusion of 1 mL normal saline at 18:00 every day.After the natural delivery of pregnant rats.10 offspring rats were randomly selected from each group.At birth,4 wk and 8 wk after the birth of offspring rats,the weight of offspring rats was measured.The blood glucose level of offspring rats was measured at 4wk and 8 wk,while the level of serum insulin,triglyceride and leptin was measured at 8 wk.Results:The weight of offspring rats at birth in the insulin group and metformin group was significantly lower than the one in the control group(P<0.05),and there was no significant difference at 4 wk and 8 wk among three groups(P>0.05).The fasting blood glucose and random blood glucose in the insulin group and metformin group at 4 wk and 8 wk were all significantly lower than ones in the control group(P<0.05);there was no significant difference between the insulin group and metformin group(P>0.05).The expression of PPARGC1 A mRNA in the insulin group and metformin group was significantly higher and the methylation level of PPARGC1 A was significantly lower than the one in the control group(P<0.05),but there was no significant difference between the insulin group and metformin group(P>0.05).Insulin and leptin at 8 wk in the insulin group and metformin group were significantly higher,while triglyceride was significantly lower than the one in the control group(P<0.05);triglyceride level of rats in the insulin group was significantly higher than the one in the metformin group(P<0.05).There was no significant difference in insulin and leptin level of offspring rats between the insulin group and metformin group(P>0.05).Conclusions:GDM can induce the methylation of PPARGC1 A of offspring rats to reduce the expression of PPARGC1 A mRNA and then cause the disorder of glycolipid metabolism when the offspring rats grow up;the insulin or metformin in the treatment of pregnant rats with GDM can reduce the methylation level of PPARGC1 A and thus improve the abnormal glycolipid metabolism of offspring rats.
基金Supported by Grants from the Italian Ministry of Education(MIUR)traveling Grants by the Italian Ministry of Foreign Affairs(to Risuleo G)
文摘AIM:To explore the possibility that PDX-1 gene is reactivated as a consequence of molecular events that occur during liver regeneration. METHODS:Rat hepatocytes were maintained in DMEM- F12,10%fetal bovine serum(FBS),penicillin/streptomycin and geneticin when applicable.Rat insulinoma RIN 1046-38 cells were maintained in M-199-10%FBS and penicillin/streptomycin.The final concentration of glucose was 11.1 mmol/L.During regeneration,lateral and medial liver lobes of adult male Wistar rats were surgically removed,with up 70%loss of liver mass.In methylation experiments,5-aza-deoxycytidine(5-aza-dC)was used.Primer3 software was used for poly- merase chain reaction(PCR).Quantitative real time PCR (qRT-PCR)was performed using SYBR Green technol- ogy;primers were designed by Beacon Designer 6 software.Western blotting and SDS-PAGE were performed according to standard procedures.Antibodies were purchased from commercial suppliers.RESULTS:We explored the possibility that liver regeneration could trigger PDX-1 expression,and hence insulin production.Twenty-four hours after surgical liver removal,regeneration was active as demonstrated by the increased proliferating cell nuclear antigen;however, all the other checked genes(involved in insulin gene expression):PC-1,Ngn3,NeuroD1,Btc,PDX-1 and Ins-1, were not related to the molecular events caused by this process.The only marker detected in regenerating liver was E47:a transcription factor of the the basic helixloop-helix family known to be expressed ubiquitously in mammalian cells.In the rat pancreas,almost all of the tested genes were expressed as shown by RT-PCR, except for Ngn3,which was silenced 2 d after birth. Therefore,the molecular events in liver regeneration are not sufficient to promote PDX-1 expression.DNA methylation is a known mechanism to achieve stable re- pression of gene expression in mammals:Hxk 2 gene is silenced through this mechanism in normal hepatocytes. The administration of 5-aza-dC to cultured cells is in fact able to upregulate Hxk 2 mRNA.We investigated whether PDX-1 silencing in liver cells could be exerted through methylation of CpG islands in both the promoter and the gene coding regions.The results show that the drug increased the expression level of the Hxk 2 control gene but failed to rescue the expression of PDX-1,thus DNA demethylation is not sufficient to override repression of the PDX-1 gene.
基金Supported by Key Project of Chinese Ministry of Science and Technology,No.2012ZX10002007 and No.2013ZX10002001National Natural Science Foundation of China,No.81171579,No.81201287 and No.81371832Science and Technology Development Plan of Shandong Province,China,No.2014GSF118068
文摘AIM: To evaluate tumor necrosis factor-α converting enzyme(TACE) methylation status in patients with chronic hepatitis B(CHB).METHODS: Eighty patients with hepatitis B e antigen(HBe Ag)-positive CHB, 80 with HBe Ag-negative CHB, and 40 healthy controls(HCs) were randomly enrolled in this study. Genomic DNA was extracted from peripheral blood mononuclear cells and methylation status of TACE promoter was determined by methylation-specific polymerase chain reaction. The clinical and laboratory parameters were collected.RESULTS: One hundred and thirty of 160 patients with CHB(81.25%) and 38 of 40 HCs(95%) displayed TACE promoter methylation. The difference was significant(χ2 = 4.501, P < 0.05). TACE promoter methylation frequency in HBe Ag-positive CHB(58/80, 72.5%) was significantly lower than that in HBe Ag-negative CHB(72/80, 90%; χ2 = 8.041, P < 0.01) and HCs(χ2 = 8.438, P < 0.01). However, no significant difference was observed in the methylation frequency between HBe Agnegative CHB and HCs(χ2 = 0.873, P > 0.05). In the HBe Ag-positive group, TACE methylation frequency was significantly negatively correlated with HBe Ag(r =-0.602, P < 0.01), alanine aminotransferase(r =-0.461, P < 0.01) and aspartate aminotransferase(r =-0.329, P < 0.01). CONCLUSION: Patients with HBe Ag-positive CHB have aberrant demethylation of the TACE promoter, which may potentially serve as a biomarker for HBe Ag seroconversion.
基金Supported by Grants-in-Aid for Scientific Research from the Ministry of Education,Culture,Sports,Science and Technology of Japan (to Yamamoto H and Shinomura Y)
文摘AIM:To evaluate the utility of measuring epigenetic alterations in pancreatic and biliary fluids in determining molecular markers for pancreatobiliary cancers.METHODS:DNA was extracted from undiluted pancreatic and biliary fluids.As a surrogate for a genomewide hypomethylation assay,levels of long interspersed nuclear element-1(LINE-1) methylation were analyzed using bisulfite pyrosequencing.CpG island hypermethylation of 10 tumor-associated genes,aryl-hydrocarbon receptor repressor,adenomatous polyposis coli,calcium channel,voltage dependent,T type α1G subunit,insulin-like growth factor 2,O-6-methyl-guanine-DNA methyltransferase,neurogenin 1,CDKN2A,runt-related transcription factor 3(RUNX3),secreted frizzled-related protein 1,and ubiquitin carboxyl-terminal esterase L1(UCHL1),was analyzed using MethyLight.To examine the role of CpG methylation and histone deacetylation in the silencing of UCHL1,human gallbladder carcinoma cell lines and pancreatic carcinoma cell lines were treated with 2 or 5 μmol/L 5-AZA-dC for 72 h or 100 nmol/L Trichostatin A for 24 h.After the treatment,UCHL1 expression was analyzed by real-time reverse transcription-polymerase chain reaction.RESULTS:Pancreatobiliary cancers exhibited significantly lower LINE-1 methylation levels in pancreatic and biliary fluids than did noncancerous pancreatobiliary disease(58.7% ± 4.3% vs 61.7% ± 2.2%,P = 0.027;53.8% ± 6.6% vs 57.5% ± 1.7%,P = 0.007);however,LINE-1 hypomethylation was more evident in pancreatic cancer tissues than in pancreatic fluids(45.4% ± 5.5% vs 58.7% ± 4.3%,P < 0.001).CpG island hypermethylation of tumor-associated genes was detected at various frequencies,but it was not correlated with LINE-1 hypomethylation.Hypermethylation of the UCHL1 gene was cancer-specific and most frequently detected in pancreatic(67%) or biliary(70%) fluids from patients with pancreatobiliary cancer.As a single marker,hypermethylation of the UCHL1 gene in pancreatic and biliary fluids was most useful for the detection of pancreatic and pancreatobiliary cancers,respectively(100% specificity).Hypermethylation of the UCHL1 and RUNX3 genes in pancreatic and biliary fluids was the most useful combined marker for pancreatic(87% sensitivity and 100% specificity) and pancreatobiliary(97% sensitivity and 100% specificity) cancers.Treatment with a demethylating agent,5-AZA-2'-deoxycytidine,restored UCHL1 expression in pancreatobiliary cancer cell lines.CONCLUSION:Our results suggest that hypermethylation of UCHL1 and RUNX3 in pancreatobiliary fluid might be useful for the diagnosis of pancreatobiliary cancers.
基金supported by the grants from the National Natural Science Foundation of China (No. 4147 6124)the Natural Science Foundation of Zhejiang Province (No. LY17C190005)+3 种基金the Major Agriculture Program of Ningbo (No. 2017C110007)the Ningbo Science and Technology Project (No. 2016C10037)the Open Fund of Ningbo University (No. xkzsc1505)K C Wong Magana Fund in Ningbo University
文摘In this study,the methylation-sensitive amplification polymorphism(MSAP)was used to compare the genomic DNA methylation level of muscle,gill and hepatopancreas of Portunus trituberculatus subjected to salinity 12 for 30 days to illustrate the epigenetic mechanism of osmoregulation.Thirty primers were used to analyze the difference of methylation level of different tissues.The results showed that the baseline methylation level of muscle,hepatopancreas and gill was 47.31%,22.94%and 17.69%,respectively.After exposed to low salinity stress,the methylation epiloci changed in the three tissues.Both demethylation and methylation processes occurred under low salinity stress.The methylation ratio decreased in muscle and gill but increased in hepatopancreas.These results indicated that DNA methylation is tissue-specific when P.trituberculatus responds to low salinity.
文摘Epigenetic changes of DNA, including methylation, have long been recognized as key indicators of various diseases, including aging, cancer, and neurological disorders. Biomarker discoveries based on distinct methylation patterns for both hypermethylation and hypomethylation lead the way in discovery of novel diagnosis and treatment targets. Many different approaches are present to detect the level of methylation in whole genome (whole genome bisulfite sequencing, microarray) as well as at specific loci (methylation specific PCR). Cell-free DNA (cf-DNA) found in body fluids like blood provides information about DNA methylation and serves as a less invasive approach for genetic screening. Cell-free DNA and methylation screening technologies, when combined, have the potential to transform the way we approach genetic screening and personalized therapy. These technologies can help enhance disease diagnostic accuracy and inform the development of targeted therapeutics by providing a non-invasive way for acquiring genomic information and identifying disease-associated methylation patterns. We highlight the clinical benefits of using cell-free DNA (cf-DNA) liquid biopsy analysis and available methylation screening technologies that have been crucial in identifying biomarkers for disease from patients using a non-invasive way. Powering such biomarker discoveries are various methods of cf-DNA methylation analysis such as Bisulfite Sequencing and most recently, Methylation-Specific Restriction Enzyme (MSRE-seq) Analysis, paving the way for novel epigenetic biomarker discoveries for more robust diagnosis such as early disease detection, prognosis, monitoring of disease progression and treatment response as well as discovery of novel drug targets.
文摘The tumor selectivity of alkylating agents that produce guanine O6-chloroethyl (laromustine and carmustine) and O6-methyl (temozolomide) lesions depends upon O6-methylguanine-DNA methyltransferase (MGMT) activity being lower in tumor than in host tissue. Despite the established role of MGMT as a tumor resistance factor, consensus on how to assess MGMT expression in clinical samples is unsettled. The aim of this study is to examine the relationship between the values derived from distinctive MGMT measurements in 13, 12, 6 and 2 pairs of human tumors and matched normal adjacent tissue from the colon, kidney, lung and liver, respectively, and in human cell lines. The MGMT measurements included 1) alkyl-transfer assays using [benzene-3H]O6-benzylguanine as a substrate to assess functional MGMT activity, 2) methylation-specific PCR (MSP) to probe MGMT gene promoter CpG methylations as a measure of gene silencing, and 3) western immunoblots to analyze the MGMT protein. In human cell lines, a strict negative correlation existed between MGMT activity and the extent of promoter methylation. In tissue specimens, by contrast, the correlation between these two variables was low. Moreover, alkyl-transfer assays identified 3 pairs of tumors and normal tissue with tumor-selective reduction in MGMT activity in the absence of promoter methylation. Cell line MGMT migrated as a single band in western analyses, whereas tissue MGMT was heterogeneous around its molecular size and at much higher molecular masses, indicative of multi-layered post-translational modifications. Malignancy is occasionally associated with a mobility shift in MGMT. Contrary to the prevalent expectation that MGMT expression is governed at the level of gene silencing, these data suggest that other mechanisms that can lead to tumorselective reduction in MGMT activity exist in human tissue.
文摘Following the publication of Xu et al.(2022),an error was identified in Figure 1D.Specifically,the top left panel was inadvertently duplicated during figure preparation.To ensure the accuracy and integrity of our published work,we request the publication of a corrigendum with the corrected image.We apologize for this oversight and any confusion it may have caused.The amended figure is provided in the updated Supplementary Materials.
文摘BACKGROUND Epigenetic regulation of leptin(LEP)plays a critical role in metabolic disorders,yet its promoter methylation patterns in lean diabetic populations remain poorly characterized.Emerging evidence suggests DNA methylation may precede clinical hyperglycemia,offering potential for early risk stratification.While obesity-associated LEP methylation is well-studied,lean Asian populations who exhibit high diabetes prevalence despite lower adiposity,represent an underexplored cohort.This study hypothesizes that LEP promoter methylation in peripheral leukocytes decreases progressively from normoglycemia to prediabetes and type 2 diabetes mellitus(T2DM),correlating inversely with serum LEP levels in lean Chinese adults[body mass index(BMI)<24 kg/m^(2)].AIM To investigate LEP promoter methylation status and its association with serum LEP levels across glycemic states in lean Chinese adults.METHODS We enrolled 392 participants including 120 normoglycemic controls,94 prediabetes[44 impaired fasting glucose(IFG)/50 impaired glucose tolerance(IGT)],178 T2DM aged 40-60 years with BMI<24 kg/m^(2).Genomic DNA from peripheral leukocytes underwent bisulfite conversion followed by methylation-specific PCR to assess CpG methylation in the LEP promoter.Serum LEP was quantified via enzyme-linked immunosorbent assay,with other parameters measured through standard assays.Statistical analyses included analysis of variance,χ²tests,and Pearson correlation(Bonferroni-corrected P value).RESULTS Methylation frequencies declined progressively:59.2%(controls)reduced to 43.6%(prediabetes;IFG:38.6%,IGT:48%)reduced to 31.5%(T2DM)(all P<0.05 vs controls;T2DM vs IGT:P=0.030).Serum LEP levels increased significantly in T2DM(16.94±4.19μg/L)vs controls(11.33±3.10μg/L;P=0.002),with intermediate values in prediabetes(IFG:13.79±3.32μg/L;IGT:12.62±4.81μg/L).A near-perfect inverse correlation between methylation and LEP levels was observed(r=-0.95,95%CI:-0.97 to-0.92,P<0.001),persisting after adjusting for age and BMI(β=-0.91,P<0.001).CONCLUSION LEP promoter hypomethylation parallels worsening glycemic status in lean Chinese adults,suggesting its potential as a blood-based epigenetic biomarker for diabetes progression,pending validation in longitudinal cohorts.
基金supported by the National Natural Science Foundation of China(32070607)the National Key Research and Development Program of China(2020YFA0804000)the CAS Project for Young Scientists in Basic Research(YSBR-012).
文摘In the mammalian genome,most CpGs are methylated.However,CpGs within the CpG islands(CGIs)are largely unmethylated,which are important for gene expression regulation.The mechanism underlying the low methylation levels at CGIs remains largely elusive.KDM2 proteins(KDM2A and KDM2B)are H3K36me2 demethylases known to bind specifically at CGIs.Here,we report that depletion of each or both KDM2 proteins,or mutation of all their JmjC domains that harbor the H3K36me2 demethylation activity,leads to an increase in DNA methylation at selective CGIs.The Kdm2a/2b double knockout shows a stronger increase in DNA methylation compared with the single mutant of Kdm2a or Kdm2b,indicating that KDM2A and KDM2B redundantly regulate DNA methylation at CGIs.In addition,the increase of CGI DNA methylation upon mutations of KDM2 proteins is associated with the chromatin environment.Our findings reveal that KDM2A and KDM2B function redundantly in regulating DNA methylation at a subset of CGIs in an H3K36me2 demethylation-dependent manner.
