Comparative genomic analysis of N6-methyladenosine regulators in nine rosaceae species and functional characterization in response to drought stress in pear

N 6-methylated adenine(m6 A)is an emerging epigenetic marker in eukaryotic organisms that plays an important role in biological functions and in enriching genetic information.m6 A exerts these functions via the dynamic interplay among m6 A writers,erasers,and readers.However,little is known about the underlying mechanisms of m6 A in plant growth and stress responses.Here,we identified 276 masked m6 A regulators from nine Rosaceae species(Pyrus bretschneideri,Pyrus betulifolia,Pyrus communis,Malus domestica,Fragaria vesca,Prunus avium,Prunus mume,Prunus persica,and Rubus occidentalis).We classified and named these genes in more detail based on phylogenetic and synteny analysis.The expansion of m6 A regulators in Maloideae was dated back to the recent whole-genome duplication(WGD)in Rosaceae.Based on the expression pattern analysis and gene structure analysis of m6 A regulators,m6 A was shown to be a significant factor in regulating plant development and resistance.In addition,PbrMTA1-silenced pear plants displayed significantly reduced drought tolerance and chlorophyll content,as well as increased electrolyte leakage and concentrations of malondialdehyde and H2 O2.

Lipid metabolism and m^(6)A RNA methylation are altered in lambs supplemented rumen-protected methionine and lysine in a low-protein diet

Background:Methionine or lysine has been reported to influence DNA methylation and fat metabolism,but their combined effects in N6-methyl-adenosine(m^(6)A)RNA methylation remain unclarified.The combined effects of rumen-protected methionine and lysine(RML)in a low-protein(LP)diet on lipid metabolism,m^(6)A RNA methylation,and fatty acid(FA)profiles in the liver and muscle of lambs were investigated.Sixty-three male lambs were divided into three treatment groups,three pens per group and seven lambs per pen.The lambs were fed a 14.5%crude protein(CP)diet(adequate protein[NP]),12.5%CP diet(LP),and a LP diet plus RML(LP+RML)for 60 d.Results:The results showed that the addition of RML in a LP diet tended to lower the concentrations of plasma leptin(P=0.07),triglyceride(P=0.05),and non-esterified FA(P=0.08).Feeding a LP diet increased the enzyme activity or m RNA expression of lipogenic enzymes and decreased lipolytic enzymes compared with the NP diet.This effect was reversed by supplementation of RML with a LP diet.The inclusion of RML in a LP diet affected the polyunsaturated fatty acids(PUFA),n-3 PUFA,and n-6 PUFA in the liver but not in the muscle,which might be linked with altered expression of FA desaturase-1(FADS1)and acetyl-Co A carboxylase(ACC).A LP diet supplemented with RML increased(P<0.05)total m^(6)A levels in the liver and muscle and were accompanied by decreased expression of fat mass and obesity-associated protein(FTO)and alk B homologue 5(ALKBH5).The m RNA expressions of methyltransferase-like 3(METTL3)and methyltransferase-like 14(METTL14)in the LP+RML diet group were lower than those in the other two groups.Supplementation of RML with a LP diet affected only liver YTH domain family(YTHDF2)proteins(P<0.05)and muscle YTHDF3(P=0.09),which can be explained by limited m^(6)Abinding proteins that were mediated in m RNA fate.Conclusions:Our findings showed that the inclusion of RML in a LP diet could alter fat deposition through modulations of lipogenesis and lipolysis in the liver and muscle.These changes in fat metabolism may be associated with the modification of m^(6)A RNA methylation.

METTL3-mediated m^(6)A RNA methylation regulates dorsal lingual epithelium homeostasis

The dorsal lingual epithelium,which is composed of taste buds and keratinocytes differentiated from K14^(+)basal cells,discriminates taste compounds and maintains the epithelial barrier.N6-methyladenosine(m^(6)A)is the most abundant mRNA modification in eukaryotic cells.How METTL3-mediated m^(6)A modification regulates K14^(+)basal cell fate during dorsal lingual epithelium formation and regeneration remains unclear.Here we show knockout of Mettl3 in K14^(+)cells reduced the taste buds and enhanced keratinocytes.Deletion of Mettl3 led to increased basal cell proliferation and decreased cell division in taste buds.Conditional Mettl3 knock-in mice showed little impact on taste buds or keratinization,but displayed increased proliferation of cells around taste buds in a protective manner during post-irradiation recovery.Mechanically,we revealed that the most frequent m^(6)A modifications were enriched in Hippo and Wnt signaling,and specific peaks were observed near the stop codons of Lats1 and FZD7.Our study elucidates that METTL3 is essential for taste bud formation and could promote the quantity recovery of taste bud after radiation.

Regulations of m^(6)A methylation on tomato fruit chilling injury

Tomato fruit are sensitive to chilling injury(CI)during cold storage.Several factors have been discovered to be involved in chilling injury of tomato fruit.Plant hormones play an important regulatory role,however,the relationship between chilling injury and N6-methyladenosine(m^(6)A)methylation of transcripts in plant hormone pathways has not been reported yet.In order to clarify the complex regulatory mechanism of m^(6)A methylation on chilling injury in tomato fruit,Nanopore direct RNA sequencing was employed.A large number of enzymes and transcription factors were found to be involved in the regulation process of fruit chilling injury,which were associated with plant hormone,such as 1-aminocyclopropane 1-carboxylate synthase(ACS),aspartate aminotransferase(AST),auxin response factor(ARF2),ethylene response factor 2(ERF2),gibberellin 20-oxidase-3(GA20ox)and jasmonic acid(JA).By conjoint analysis of the differential expression transcripts related to chilling injury andm^(6)Amethylation differential expression transcripts 41 differential expression transcripts were identified involved in chilling injury including 1-aminocyclopropane-1-carboxylate oxidase(ACO)and pectinesterase(PE)were down-regulated and heat shock cognate 70 kD protein 2(cpHSC70),HSP70-binding protein(HspBP)and salicylic acid-binding protein 2(SABP2)were up-regulated.Our results will provide a deeper understanding for chilling injury regulatory mechanism and post-harvest cold storage of tomato fruit.

Prognostic Evaluation for Oral Squamous Cell Carcinoma:A Novel Method Based on m6A Methylation Regulators

Objective:This study aimed to examine a novel method for prognostic evaluation of patients with oral squamous cell carcinoma(OSCC)based on the expression of heterogeneous nuclear ribonucleoprotein C(HNRNPC),YTH domain-binding protein 2(YTHDF2),and methyltransferase 14(METTL14).Methods:We obtained the RNA sequence and clinical information of OSCC patients from The Cancer Genome Atlas database.An optical method was established by the least absolute shrinkage and selection operator Cox regression algorithm,which was used to calculate the risk score of every sample.In addition,all samples(n=239)were classified into high-risk(n=119)and low-risk(n=120)groups,and the overall survival(OS)time and clinical characteristics were compared between groups.Moreover,bioinformatics analysis was carried out.Gene set enrichment analysis was performed to investigate the signaling pathways of HNRNPC,YTHDF2,and METTL14.Results:The two groups showed significantly different OS time,tumor grades,tumor stages,and pathologic T stages(P<0.05).The receiver operating characteristic analysis identified that our method was effective and it was more accurate than use of age,gender,tumor grade,tumor stage,pathologic T stage,and pathologic N stage in OSCC prognostic prediction.Gene set enrichment analysis revealed that HNRNPC,YTHDF2,and METTL14 were mainly associated with ubiquitin-mediated proteolysis,cell cycle,RNA degradation,and spliceosome signaling pathways.Conclusion:The method based on the expression of HNRNPC,YTHDF2,and METTL14 can predict the prognosis of patients with OSCC independently,and its prognostic value is better than that of clinicopathological characteristic indicators.

New insights into developmental biology of Eimeria tenella revealed by comparative analysis of mRNA N6-methyladenosine modification between unsporulated oocysts and sporulated oocysts

Evidence showed that N6-methyladenosine(m^(6)A)modification plays a pivotal role in influencing RNA fate and is strongly associated with cell growth and developmental processes in many species.However,no information regarding m^(6)A modification in Eimeria tenella is currently available.In the present study,we surveyed the transcriptome-wide prevalence of m^(6)A in sporulated oocysts and unsporulated oocysts of E.tenella.Methylated RNA immunoprecipitation sequencing(MeRIP-seq)analysis showed that m^(6)A modification was most abundant in the coding sequences,followed by stop codon.There were 3,903 hypermethylated and 3,178 hypomethylated mRNAs in sporulated oocysts compared with unsporulated oocysts.Further joint analysis suggested that m^(6)A modification of the majority of genes was positively correlated with mRNA expression.The mRNA relative expression and m^(6)A level of the selected genes were confirmed by quantitative reverse transcription PCR(RT-qPCR)and MeRIP-qPCR.GO and KEGG analysis indicated that differentially m^(6)A methylated genes(DMMGs)with significant differences in mRNA expression were closely related to processes such as regulation of gene expression,epigenetic,microtubule,autophagy-other and TOR signaling.Moreover,a total of 96 DMMGs without significant differences in mRNA expression showed significant differences at protein level.GO and pathway enrichment analysis of the 96 genes showed that RNA methylation may be involved in cell biosynthesis and metabolism of E.tenella.We firstly present a map of RNA m^(6)A modification in E.tenella,which provides significant insights into developmental biology of E.tenella.

Enhancing m^(6)A modification in the motor cortex facilitates corticospinal tract remodeling after spinal cord injury

Spinal cord injury typically causes corticospinal tract disruption. Although the disrupted corticospinal tract can self-regenerate to a certain degree, the underlying mechanism of this process is still unclear. N6-methyladenosine(m^(6)A) modifications are the most common form of epigenetic regulation at the RNA level and play an essential role in biological processes. However, whether m^(6)A modifications participate in corticospinal tract regeneration after spinal cord injury remains unknown. We found that expression of methyltransferase 14 protein(METTL14) in the locomotor cortex was high after spinal cord injury and accompanied by elevated m^(6)A levels. Knockdown of Mettl14 in the locomotor cortex was not favorable for corticospinal tract regeneration and neurological recovery after spinal cord injury. Through bioinformatics analysis and methylated RNA immunoprecipitation-quantitative polymerase chain reaction, we found that METTL14 regulated Trib2 expression in an m^(6)A-regulated manner, thereby activating the mitogen-activated protein kinase pathway and promoting corticospinal tract regeneration. Finally, we administered syringin, a stabilizer of METTL14, using molecular docking. Results confirmed that syringin can promote corticospinal tract regeneration and facilitate neurological recovery by stabilizing METTL14. Findings from this study reveal that m^(6)A modification is involved in the regulation of corticospinal tract regeneration after spinal cord injury.

O6-methylguanine DNA methyltransferase is upregulated in malignant transformation of gastric epithelial cells via its gene promoter DNA hypomethylation

BACKGROUND O_(6)-methylguanine-DNA methyltransferase(MGMT)is a suicide enzyme that repairs the mispairing base O_(6)-methyl-guanine induced by environmental and experimental carcinogens.It can transfer the alkyl group to a cysteine residue in its active site and became inactive.The chemical carcinogen N-nitroso compounds(NOCs)can directly bind to the DNA and induce the O_(6)-methylguanine adducts,which is an important cause of gene mutation and tumorigenesis.However,the underlying regulatory mechanism of MGMT involved in NOCs-induced tumorigenesis,especially in the initiation phase,remains largely unclear.AIM To investigate the molecular regulatory mechanism of MGMT in NOCs-induced gastric cell malignant transformation and tumorigenesis.METHODS We established a gastric epithelial cell malignant transformation model induced by N-methyl-N’-nitro-N-nitrosoguanidine(MNNG)or N-methyl-N-nitroso-urea(MNU)treatment.Cell proliferation,colony formation,soft agar,cell migration,and xenograft assays were used to verify the malignant phenotype.By using quantitative real-time polymerase chain reaction(qPCR)and Western blot analysis,we detected the MGMT expression in malignant transformed cells.We also confirmed the MGMT expression in early stage gastric tumor tissues by qPCR and immunohistochemistry.MGMT gene promoter DNA methylation level was analyzed by methylation-specific PCR and bisulfite sequencing PCR.The role of MGMT in cell malignant transformation was analyzed by colony formation and soft agar assays.RESULTS We observed a constant increase in MGMT mRNA and protein expression in gastric epithelial cell malignant transformation induced by MNNG or MNU treatment.Moreover,we found a reduction of MGMT gene promoter methylation level by methylation-specific PCR and bisulfite sequencing PCR in MNNG/MNU-treated cells.Inhibition of the MGMT expression by O_(6)-benzylguanine promoted the MNNG/MNU-induced malignant phenotypes.Overexpression of MGMT partially reversed the cell malignant transformation process induced by MNNG/MNU.Clinical gastric tissue analysis showed that MGMT was upregulated in the precancerous lesions and metaplasia tissues,but downregulated in the gastric cancer tissues.CONCLUSION Our finding indicated that MGMT upregulation is induced via its DNA promoter hypomethylation.The highly expressed MGMT prevents the NOCs-induced cell malignant transformation and tumorigenesis,which suggests a potential novel approach for chemical carcinogenesis intervention by regulating aberrant epigenetic mechanisms.

m^(6)A RNA甲基化调节因子与前列腺癌预后的关系

目的探索N6-甲基腺嘌呤(m^(6)A)RNA甲基化调节因子(以下简称m^(6)A调节因子)与前列腺癌预后的关系。方法从癌症基因组图谱(TCGA)数据库下载416例前列腺癌样本和80例癌旁样本的临床病理数据及其mRNA相关数据,获取METTL3、METTL14、WTAP、RBM15、ZC3H13、YTHDC1、YTHDC2、YTHDF1、YTHDF2、HNRNPC、FTO和ALKBH5共12种m^(6)A调节因子。筛选出前列腺癌样本差异表达的m^(6)A调节因子。对前列腺癌组织样本进行无监督聚类分组,并比较其总体生存率的差异。进行多因素Cox回归分析,根据风险评分分为高风险组和低风险组,比较2组生存率。对临床病理因素进行风险评分,构建多因素Cox回归模型,评估预后预测价值。采用免疫组织化学染色方法检测前列腺癌组织中METTL14和FTO的表达。结果从12个m^(6)A调节因子中筛选出8个差异表达的调节因子。通过无监督聚类分析将前列腺癌样本分成3组,分别为Cluster 1、Cluster 2和Cluster 3,3组的生存时间存在显著差异。多因素Cox回归分析发现,METTL14和FTO与前列腺癌患者的预后密切相关。构建Cox回归模型,对前列腺癌患者进行风险评分,发现高、低风险组总生存率有统计学差异,且风险评分可作为独立预后指标。前列腺癌组织中METTL14和FTO蛋白阳性表达率明显高于癌旁组织(P<0.05)。结论本研究构建了基于m^(6)A调节因子的前列腺癌预后预测模型,其中风险评分可作为独立的预后指标,METTL14和FTO可作为诊断前列腺癌的分子标志物和治疗的潜在靶点。

The role of m^(6)A RNA methylation in autoimmune diseases: Novel therapeutic opportunities

N6-methyladenosine m^(6)A modifications,as one of the most common forms of in-ternal RNA chemical modifications in eukaryotic cells,have gained increasing attention in recent years.The m^(6)A RNA modifications exert various crucial roles in various biological pro-cesses,such as embryonic development,neurogenesis,circadian rhythms,and tumorigenesis.Recent advances have highlighted that m^(6)A RNA modification plays an important role in im-mune response,especially in the initiation and progression of autoimmune diseases.In this re-view,we summarized the regulatory mechanisms of m^(6)A methylation and its biological functions in the immune system and mainly focused on recent progress in research on the po-tential role of m^(6)A RNA methylation in the pathogenesis of autoimmune diseases,thus providing possible biomarkers and potential targets for the prevention and treatment of auto-immunediseases.

The regulatory role of m^(6)A modification in the maintenance and differentiation of embryonic stem cells

As the most prevalent and reversible internal epigenetic modification in eukaryotic mRNAs,N6-methyladenosine(m^(6)A)post-transcriptionally regulates the processing and metabolism of mRNAs involved in diverse biological processes.m^(6)A modification is regulated by m^(6)A writers,erasers,and readers.Emerging evidence suggests that m^(6)A modification plays essential roles in modulating the cell-fate transition of embryonic stem cells.Mechanistic investigation of embryonic stem cell maintenance and differentiation is critical for understanding early embryonic development,which is also the premise for the application of embryonic stem cells in regenerative medicine.This review highlights the current knowledge of m^(6)A modification and its essential regulatory contribution to the cell fate transition of mouse and human embryonic stem cells.

Dynamic RNA methylation modifications and their regulatory role in mammalian development and diseases

Among over 170 different types of chemical modifications on RNA nucleobases identified so far,RNA methylation is the major type of epitranscriptomic modifications existing on almost all types of RNAs,and has been demonstrated to participate in the entire process of RNA metabolism,including transcription,pre-mRNA alternative splicing and maturation,mRNA nucleus export,mRNA degradation and stabilization,mRNA translation.Attributing to the development of high-throughput detection technologies and the identification of both dynamic regulators and recognition proteins,mechanisms of RNA methylation modification in regulating the normal development of the organism as well as various disease occurrence and developmental abnormalities upon RNA methylation dysregulation have become increasingly clear.Here,we particularly focus on three types of RNA methylations:N^(6)-methylcytosine(m^(6)A),5-methylcytosine(m^(5)C),and N^(7)-methyladenosine(m^(7)G).We summarize the elements related to their dynamic installment and removal,specific binding proteins,and the development of high-throughput detection technologies.Then,for a comprehensive understanding of their biological significance,we also overview the latest knowledge on the underlying mechanisms and key roles of these three mRNA methylation modifications in gametogenesis,embryonic development,immune system development,as well as disease and tumor progression.

Regulation of m^(6)Am RNA modification and its implications in human diseases

N6,2′-O-dimethyladenosine(m^(6)Am)is a prevalent modification frequently found at the 5′cap-adjacent adenosine of messenger RNAs(mRNAs)and small nuclear RNAs(snRNAs)and the internal adenosine of snRNAs.This dynamic and reversible modification is under the regulation of methyltransferases phosphorylated CTD interacting factor 1 and methyltransferase-like protein 4,along with the demethylase fat mass and obesity-associated protein.m^(6)Am RNA modification plays a crucial role in the regulation of pre-mRNA splicing,mRNA stability,and translation,thereby influencing gene expression.In recent years,there has been growing interest in exploring the functions of m^(6)Am and its relevance to human diseases.In this review,we provide a comprehensive overview of the current knowledge concerning m^(6)Am,with a focus on m^(6)Am-modifying enzymes,sequencing approaches for its detection,and its impacts on pre-mRNA splicing,mRNA stability,and translation regulation.Furthermore,we highlight the roles of m^(6)Am in the context of obesity,viral infections,and cancers,unravelling its underlying regulatory mechanisms.

The landscape of epigenetic regulation and therapeutic application of N^(6)-methyladenosine modifications in non-coding RNAs

RNA N^(6)-methyladenosine(m^(6)A)methylation is the most abundant and conserved RNA modification in eukaryotes.It participates in the regulation of RNA metabolism and various pathophysiological processes.Non-coding RNAs(ncRNAs)are defined as small or long transcripts which do not encode proteins and display numerous biological regulatory functions.Similar to mRNAs,m^(6)A deposition is observed in ncRNAs.Studying RNA m^(6)A modifications on ncRNAs is of great importance specifically to deepen our understanding of their biological roles and clinical implications.In this review,we summarized the recent research findings regarding the mutual regulation between RNA m^(6)A modification and ncRNAs(with a specific focus on microRNAs,long non-coding RNAs,and circular RNAs)and their functions.We also discussed the challenges of m^(6)A-containing ncRNAs and RNA m^(6)A as therapeutic targets in human diseases and their future perspective in translational roles.

m^(6)A RNA甲基化调节因子与肝细胞癌预后的关系

目的探讨N^(6)-甲基腺嘌呤(m^(6)A)RNA甲基化调节因子与肝细胞癌(HCC)预后的关系。方法从美国国家癌症研究所基因组数据共享中心下载HCC样本和正常对照样本的RNA-seq转录组数据,并收集HCC样本临床病理资料。从RNA-seq转录组数据中获取FTO、YTHDC2、YTHDC1、ALKBH5、KIAA1429、METTL3、HNRNPC、YTHDF2、RBM15、YTHDF1、WTAP、METTL14、ZC3H13等十三种m^(6)A RNA甲基化调节因子的表达数据,比较HCC样本和正常对照样本m^(6)A RNA甲基化调节因子表达。采用单因素Cox回归分析和Lasso Cox回归分析筛选与HCC预后相关的m^(6)A RNA甲基化调节因子,并构建风险模型,分析风险模型与HCC预后的关系。结果 HCC样本FTO、YTHDC2、YTHDC1、ALKBH5、KIAA1429、METTL3、HNRNPC、YTHDF2、RBM15、YTHDF1、WTAP表达均高于正常对照样本(P均<0.01),而两样本METTL14、ZC3H13表达比较差异均无统计学意义(P均>0.05)。YTHDF1、ZC3H13、YTHDF2、METTL3和KIAA1429等五种m^(6)A RNA甲基化调节因子是影响HCC预后的危险因素(P均<0.05)。其中,ZC3H13高表达者预后良好,而YTHDF1、YTHDF2、METTL3、KIAA1429高表达者预后较差。根据YTHDF1、ZC3H13、YTHDF2、METTL3、KIAA1429等五种m^(6)A RNA甲基化调节因子构建HCC预后风险模型,然后将HCC患者分为高风险者与低风险者,高风险者5年生存率明显低于低风险者(P<0.05);ROC曲线分析显示,该风险模型的曲线下面积为0.619。单因素和多因素Cox回归分析显示,风险模型评分是影响HCC预后的独立危险因素(P<0.01)。结论 m^(6)A RNA甲基化调节因子表达与HCC预后密切相关,有可能作为肿瘤诊断的分子标志物和潜在的治疗靶点。

Coordinated transcriptional and post-transcriptional epigenetic regulation during skeletal muscle development and growth in pigs

Background:N6-methyladenosine(m^(6)A)and DNA 5-methylcytosine(5mC)methylation plays crucial roles in diverse biological processes,including skeletal muscle development and growth.Recent studies unveiled a potential link between these two systems,implicating the potential mechanism of coordinated transcriptional and post-transcrip-tional regulation in porcine prenatal myogenesis and postnatal skeletal muscle growth.Methods:Immunofluorescence and co-IP assays were carried out between the 5mC writers and m^(6)A writers to investigate the molecular basis underneath.Large-scale in-house transcriptomic data were compiled for applying weighted correlation network analysis(WGCNA)to identify the co-expression patterns of m^(6)A and 5mC regulators and their potential role in pig myogenesis.Whole-genome bisulfite sequencing(WGBS)and methylated RNA immu-noprecipitation sequencing(MeRIP-seq)were performed on the skeletal muscle samples from Landrace pigs at four postnatal growth stages(days 30,60,120 and 180).Results:Significantly correlated expression between 5mC writers and m^(6)A writers and co-occurrence of 5mC and m^(6)A modification were revealed from public datasets of C2C12 myoblasts.The protein-protein interactions between the DNA methylase and the m^(6)A methylase were observed in mouse myoblast cells.Further,by analyzing tran-scriptome data comprising 81 pig skeletal muscle samples across 27 developmental stages,we identified a 5mC/m^(6)A epigenetic module eigengene and decoded its potential functions in pre-or post-transcriptional regulation in postnatal skeletal muscle development and growth of pigs.Following integrative multi-omics analyses on the WGBS methylome data and MeRIP-seq data for both m^(6)A and gene expression profiles revealed a genome/transcriptome-wide correlated dynamics and co-occurrence of 5mC and m^(6)A modifications as a consequence of 5mC/m^(6)A crosstalk in the postnatal myogenesis progress of pigs.Last,we identified a group of myogenesis-related genes collaboratively regulated by both 5mC and m^(6)A modifications in postnatal skeletal muscle growth in pigs.Conclusions:Our study discloses a potential epigenetic mechanism in skeletal muscle development and provides a novel direction for animal breeding and drug development of related human muscle-related diseases.

miR-6803和PPP6R1在食管鳞状细胞癌组织和细胞中的表达及其甲基化状态与患者临床病理特征的关系

目的:探讨微小RNA-6803(mi R-6803)及其宿主基因蛋白磷酸酶6调节亚单位1(protein phosphatase 6 regulation subunit 1,PPP6R1)在食管鳞状细胞癌(ESCC)中的表达和PPP6R1基因启动子区甲基化状态及其在ESCC发生及发展中的作用。方法:采用2013年至2014年间河北医科大学第四医院生物标本库的72例ESCC手术患者癌组织及对应的癌旁组织标本,用实时荧光定量PCR法检测mi R-6803和PPP6R1在ESCC组织及其癌旁组织和DNA甲基化转移酶抑制剂5-氮杂-2’-脱氧胞苷(5-Aza-d C)处理前后的ESCC细胞株TE1、TE13、Eca109、T.TN、Kyse170中的表达水平。用甲基化特异性PCR(MSP)法检测ESCC细胞系和组织及其癌旁组织中PPP6R1的甲基化状态,分析其与患者临床病理特征的关系。结果:ESCC组织中mi R-6803和PPP6R1的表达水平显著低于癌旁组织(0.318±0.156,0.408±0.177 vs 1.000±0.001,均P<0.05),mi R-6803表达水平与淋巴结转移、组织分化程度及TNM分期密切相关(均P<0.05);PPP6R1表达水平与TNM分期和组织学分化程度密切相关(均P<0.05)。ESCC组织中mi R-6803和PPP6R1基因的表达具有显著相关性(P<0.05)。ESCC组织中PPP6R1的启动子区甲基化率显著高于癌旁组织(56.94%vs 36.11%,P<0.05),并与TNM分期和组织学分化程度密切相关(均P<0.05),mi R-6803和PPP6R1基因的低表达与PPP6R1启动子区甲基化明显相关(P<0.05)。经5-Aza-d C处理后,5种ESCC细胞中mi R-6803和PPP6R1的表达均升高,并且TE1、TE13、Kyse170细胞中PPP6R1基因甲基化程度降低,非甲基化程度增加,其余2种细胞中PPP6R1基因均表现为非甲基化状态。结论:mi R-6803及其宿主基因PPP6R1的低表达可能与ESCC的发生发展密切相关,其启动子区甲基化可能是mi R-6803和PPP6R1表达沉默的机制之一。

白介素-6保护小脑颗粒神经元抵抗NMDA的神经毒性作用

近来研究发现,细胞因子白介素-6(interleukin-6,IL-6)不仪是重要的免疫调节因子,而且也是重要的神经调节因子。中枢神经系统中IL-6的作用是复杂的,尤其是对脑损伤时IL-6所发挥的作用及其作用机制尚不十分清楚。为此,我们利用N-甲基-D-天门冬氨酸(N-methyl-D-aspartate,NMDA)处理小脑颗粒神经元引起急性损伤的细胞模型,探讨IL-6对神经元损伤的保护及其作用机制。取出生后8 d幼鼠的小脑,进行小脑颗粒神经元培养。将IL-6(5或10 ng/mL)加入到小脑颗粒神经元的培养液中孵育8 d,然后用NMDA(100μmol／L)刺激小脑颗粒神经元30 min以造成神经元损伤。用噻唑兰(methyl-thiazole tetrazolium,MTT)比色法检测神经元的活性;用末端脱氧核苷酸转移酶介导的原位缺口末端标记(terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling,TUNEL)法检测神经元的凋亡;用激光扫描共聚焦显微镜(confocal laser scanning microscope,CLSM)观察神经元内游离Ca2+浓度的动态变化。用抗gp130单克隆抗体(75 ng／mL)抑制IL-6的活性,然后观察IL-6抗NMDA神经毒性作用的变化;并利用Western blot法检测IL-6对小脑颗粒神经元表达IL-6的细胞内信号转导蛋白——信号转导子和转录激活子3(signal transducer and activator of transcription 3,STAT3)和细胞外信号调节激酶(extracellular signal regulated kinase 1／2,ERK1／2)磷酸化水平的影响。NMDA刺激未经IL-6处理的小脑颗粒神经元,导致神经元活性降低、神经元凋亡增加和神经元内Ca2+超载。NMDA刺激经IL-6慢性预处理的小脑颗粒神经元,其损伤程度与未经IL-6处理的神经元相比明显减轻,包括神经元活性明显增强、神经元凋亡显著减少和神经元内Ca2+超载减轻。抗gp130抗体可阻断IL-6减轻NMDA激发的神经元内Ca2+超载的作用;经IL-6慢性处理的小脑颗粒神经元,其细胞内STAT3和ERK1／2的磷酸化水平显著增加。结果表明,IL-6能保护神经元抵抗由NMDA诱导的兴奋性神经毒性,此神经保护机制与IL-6减轻神经元内Ca2+超载密切相关,而且可能通过激活神经元内IL-6的信号转导路径调节细胞内的基因表达而实现。

Ferroptosis:a critical mechanism of N^(6)-methyladenosine modification involved in carcinogenesis and tumor progression

Ferroptosis is an iron-dependent regulatory cell necrosis induced by iron overload and lipid peroxidation.It occurs when multiple redoxactive enzymes are ectopically expressed or show abnormal function.Hence,the precise regulation of ferroptosis-related molecules is mediated across multiple levels,including transcriptional,posttranscriptional,translational,and epigenetic levels.N^(6)-methyladenosine(m^(6)A)is a highly evolutionarily conserved epigenetic modification in mammals.The m^(6)A modification is commonly linked to tumor proliferation,progression,and therapy resistance because it is involved in RNA metabolic processes.Intriguingly,accumulating evidence suggests that dysregulated ferroptosis caused by the m^(6)A modification drives tumor development.In this review,we summarized the roles of m^(6)A regulators in ferroptosis-mediated malignant tumor progression and outlined the m^(6)A regulatory mechanism involved in ferroptosis pathways.We also analyzed the potential value and application strategies of targeting m^(6)A/ferroptosis pathway in the clinical diagnosis and therapy of tumors.

FLK is an mRNA m^(6)A reader that regulates floral transition by modulating the stability and splicing of FLC in Arabidopsis

N^(6)-methyladenosine(m^(6)A),which is added,removed,and interpreted by m^(6)A writers,erasers,and readers,respectively,is the most abundant modification in eukaryotic mRNAs.The m^(6)A marks play a pivotal role in the regulation of floral transition in plants.FLOWERING LOCUS K(FLK),an RNA-binding protein harboring K-homology(KH)motifs,is known to regulate floral transition by repressing the levels of a key floral repressor FLOWERING LOCUS C(FLC)in Arabidopsis.However,the molecular mechanism underlying FLK-mediated FLC regulation remains unclear.In this study,we identified FLK as a novel mRNA m^(6)A reader protein that directly binds the m^(6)A site in the 3ʹ-untranslated region of FLC transcripts to repressing FLC levels by reducing its stability and splicing.Importantly,FLK binding of FLC transcripts was abolished in vir-1,an m^(6)A writer mutant,and the late-flowering phenotype of the flk mutant could not be rescued by genetic complementation using the mutant FLKm gene,in which the m^(6)A reader encoding function was eliminated,indicating that FLK binds and regulates FLC expression in an m^(6)A-dependent manner.Collectively,our study has addressed a long-standing question of how FLK regulates FLC transcript levels and established a molecular link between the FLK-mediated recognition of m^(6)A modifications on FLC transcripts and floral transition in Arabidopsis.