Learning Sequential and Structural Dependencies Between Nucleotides for RNA N6-Methyladenosine Site Identification

N6-methyladenosine(m6A)is an important RNA methylation modification involved in regulating diverse biological processes across multiple species.Hence,the identification of m6A modification sites provides valuable insight into the biological mechanisms of complex diseases at the post-transcriptional level.Although a variety of identification algorithms have been proposed recently,most of them capture the features of m6A modification sites by focusing on the sequential dependencies of nucleotides at different positions in RNA sequences,while ignoring the structural dependencies of nucleotides in their threedimensional structures.To overcome this issue,we propose a cross-species end-to-end deep learning model,namely CR-NSSD,which conduct a cross-domain representation learning process integrating nucleotide structural and sequential dependencies for RNA m6A site identification.Specifically,CR-NSSD first obtains the pre-coded representations of RNA sequences by incorporating the position information into single-nucleotide states with chaos game representation theory.It then constructs a crossdomain reconstruction encoder to learn the sequential and structural dependencies between nucleotides.By minimizing the reconstruction and binary cross-entropy losses,CR-NSSD is trained to complete the task of m6A site identification.Extensive experiments have demonstrated the promising performance of CR-NSSD by comparing it with several state-of-the-art m6A identification algorithms.Moreover,the results of cross-species prediction indicate that the integration of sequential and structural dependencies allows CR-NSSD to capture general features of m6A modification sites among different species,thus improving the accuracy of cross-species identification.

Characterization of N6-methyladenosine long non-coding RNAs in sporadic congenital cataract and age-related cataract

AIM:To characterize the N6-methyladenosine(m6A)modification patterns in long non-coding RNAs(lncRNAs)in sporadic congenital cataract(CC)and age-related cataract(ARC).METHODS:Anterior capsule of the lens were collected from patients with CC and ARC.Methylated RNA immunoprecipitation with next-generation sequencing and RNA sequencing were performed to identify m6A-tagged lncRNAs and lncRNAs expression.Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses and Gene Ontology annotation were used to predict potential functions of the m6A-lncRNAs.RESULTS:Large amount of m6A peaks within lncRNA were identified for both CC and ARC,while the level was much higher in ARC(49870 peaks)than that in CC(18688 peaks),yet those difference between ARC in younger age group(ARC-1)and ARC in elder age group(ARC-2)was quite slight.A total of 1305 hypermethylated and 1178 hypomethylated lncRNAs,as well as 182 differential expressed lncRNAs were exhibited in ARC compared with CC.On the other hand,5893 hypermethylated and 5213 hypomethylated lncRNAs,as well as 155 significantly altered lncRNA were identified in ARC-2 compared with ARC-1.Altered lncRNAs in ARC were mainly associated with the organization and biogenesis of intracellular organelles,as well as nucleotide excision repair.CONCLUSION:Our results for the first time present an overview of the m6A methylomes of lncRNA in CC and ARC,providing a solid basis and uncovering a new insight to reveal the potential pathogenic mechanism of CC and ARC.

ALKBH5 suppresses autophagic flux via N6-methyladenosine demethylation of ZKSCAN3 mRNA in acute pancreatitis

BACKGROUND Increasing evidence has demonstrated that N6-methyladenosine(m6A)RNA modification plays an essential role in a wide range of pathological conditions.Impaired autophagy is a critical hallmark of acute pancreatitis(AP).AIM To explore the role of the m6A modification of ZKSCAN3 in the regulation of autophagy in AP.METHODS The AP mouse cell model was established by cerulein-treated mouse pancreatic acinar cells(MPC-83),and the results were confirmed by the levels of amylase and inflammatory factors.Autophagy activity was evaluated by specific identification of the autophagy-related microstructure and the expression of autophagy-related genes.ZKSCAN3 and ALKBH5 were knocked down to study the function in AP.A m6A RNA binding protein immunoprecipitation assay was used to study how the m6A modification of ZKSCAN3 mRNA is regulated by ALKBH.RESULTS The increased expression of amylase and inflammatory factors in the supernatant and the accumulation of autophagic vacuoles verified that the AP mouse cell model was established.The downregulation of LAMP2 and upregulation of LC3-II/I and SQSTM1 demonstrated that autophagy was impaired in AP.The expression of ZKSCAN3 was upregulated in AP.Inhibition of ZKSCAN3 increased the expression of LAMP2 and decreased the expression of the inflammatory factors,LC3-II/I and SQSTM1.Furthermore,ALKBH5 was upregulated in AP.Knockdown of ALKBH5 downregulated ZKSCAN3 expression and restored decreased autophagic flux in AP.Notably,the bioinformatic analysis revealed 23 potential m6A modification sites on ZKSCAN3 mRNA.The m6A modification of ZKSCAN3 mRNA was significantly decreased in AP.Knockdown of ALKBH5 increased the modification of ZKSCAN3 mRNA,which confirmed that ALKBH5 upregulated ZKSCAN3 expression in a m6A-dependent manner.CONCLUSION ALKBH5 inhibits autophagic flux through m6A demethylation of ZKSCAN3 mRNA in AP,thereby aggravating the severity of the disease.

Modulation of host N6-methyladenosine modification by gut microbiota in colorectal cancer

As a research hotspot in the field of molecular biology,N6-methyladenosine(m6A)modification has made progress in the treatment of colorectal cancer(CRC),leukemia and other cancers.Numerous studies have demonstrated that the tumour microenvironment(TME)regulates the level of m6A modification in the host and activates a series of complex epigenetic signalling pathways through interactions with CRC cells,thus affecting the progression and prognosis of CRC.However,with the diversity in the composition of TME factors,this action is reci-procal and complex.Encouragingly,some studies have experimentally revealed that the intestinal flora can alter CRC cell proliferation by directly acting on m6A and thereby altering CRC cell proliferation.This review summarizes the data,supporting the idea that the intestinal flora can influence host m6A levels through pathways such as methyl donor metabolism and thus affect the progression of CRC.We also review the role of m6A modification in the diagnosis,treatment,and prognostic assessment of CRC and discuss the current status,limitations,and potential clinical value of m6A modification in this field.We propose that additional in-depth research on m6A alterations in CRC patients and their TME-related targeted therapeutic issues will lead to better therapeutic outcomes for CRC patients.

N6-methyladenosine methylation regulates the tumor microenvironment of Epstein-Barr virus-associated gastric cancer

BACKGROUND N6-methyladenosine(m6A)methylation modification exists in Epstein-Barr virus(EBV)primary infection,latency,and lytic reactivation.It also modifies EBV latent genes and lytic genes.EBV-associated gastric cancer(EBVaGC)is a distinctive molecular subtype of GC.We hypothesized EBV and m6A methylation regulators interact with each other in EBVaGC to differentiate it from other types of GC.AIM To investigate the mechanisms of m6A methylation regulators in EBVaGC to determine the differentiating factors from other types of GC.METHODS First,The Cancer Gene Atlas and Gene Expression Omnibus databases were used to analyze the expression pattern of m6A methylation regulators between EBVaGC and EBV-negative GC(EBVnGC).Second,we identified Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)functional enrichment of m6A-related differentially expressed genes.We quantified the relative abundance of immune cells and inflammatory factors in the tumor microenvironment(TME).Finally,cell counting kit-8 cell proliferation test,transwell test,and flow cytometry were used to verify the effect of insulin-like growth factor binding protein 1(IGFBP1)in EBVaGC cell lines.RESULTS m6A methylation regulators were involved in the occurrence and development of EBVaGC.Compared with EBVnGC,the expression levels of m6A methylation regulators Wilms tumor 1-associated protein,RNA binding motif protein 15B,CBL proto-oncogene like 1,leucine rich pentatricopeptide repeat containing,heterogeneous nuclear ribonucleoprotein A2B1,IGFBP1,and insulin-like growth factor 2 binding protein 1 were significantly downregulated in EBVaGC(P<0.05).The overall survival rate of EBVaGC patients with a lower expression level of IGFBP1 was significantly higher(P=0.046).GO and KEGG functional enrichment analyses showed that the immunity pathways were significantly activated and rich in immune cell infiltration in EBVaGC.Compared with EBVnGC,the infiltration of activated CD4+T cells,activated CD8+T cells,monocytes,activated dendritic cells,and plasmacytoid dendritic cells were significantly upregulated in EBVaGC(P<0.001).In EBVaGC,the expression level of proinflammatory factors interleukin(IL)-17,IL-21,and interferon-γ and immunosuppressive factor IL-10 were significantly increased(P<0.05).In vitro experiments demonstrated that the expression level of IGFBP1 was significantly lower in an EBVaGC cell line(SNU719)than in an EBVnGC cell line(AGS)(P<0.05).IGFBP1 overexpression significantly attenuated proliferation and migration and promoted the apoptosis levels in SNU719.Interfering IGFBP1 significantly promoted proliferation and migration and attenuated the apoptosis levels in AGS.CONCLUSION m6A regulators could remodel the TME of EBVaGC,which is classified as an immune-inflamed phenotype and referred to as a“hot”tumor.Among these regulators,we demonstrated that IGFBP1 affected proliferation,migration,and apoptosis.

Advances of N6-methyladenosine modification on circular RNA in hepatocellular carcinoma

N6-methyladenosine(m6A)is a reversible epigenetic modification, which is one of the most abundant modifiers in eukaryotic cells and has been commonly reported in messenger RNAs and non-coding RNAs. The processing modification of m6A regulates RNA transcription, processing, splicing, degradation, and translation, and plays an important role in the biological process of tumors. Circular RNA, which lacks the 5' cap structure, has been mistakenly regarded as a "junk sequence" generated by accidental shearing during the transcription process. However, it has been found that circRNAs can be involved in tumor invasion and metastasis through microRNAs, binding proteins, translated peptides, and m6A modifications. In this paper, we reviewed the role of m6A modifications in circRNA regulation and their functions in hepatocellular carcinoma and discussed their potential clinical applications and future development in this field.

Long non-coding RNA GATA6-AS1 is mediated by N6-methyladenosine methylation and inhibits the proliferation and metastasis of gastric cancer

BACKGROUND Through experimental research on the biological function of GATA6-AS1,it was confirmed that GATA6-AS1 can inhibit the proliferation,invasion,and migration of gastric cancer cells,suggesting that GATA6-AS1 plays a role as an anti-oncogene in the occurrence and development of gastric cancer.Further experi-ments confirmed that the overexpression of fat mass and obesity-associated protein(FTO)inhibited the expression of GATA6-AS1,thereby promoting the occurrence and development of gastric cancer.AIM To investigate the effects of GATA6-AS1 on the proliferation,invasion and migration of gastric cancer cells and its mechanism of action.METHODS We used bioinformatics methods to analyze the Cancer Genome Atlas(https://portal.gdc.cancer.gov/.The Cancer Genome Atlas)and download expression data for GATA6-AS1 in gastric cancer tissue and normal tissue.We also constructed a GATA6-AS1 lentivirus overexpression vector which was transfected into gastric cancer cells to investigate its effects on proliferation,migration and invasion,and thereby clarify the expression of GATA6-AS1 in gastric cancer and its biological role in the genesis and development of gastric cancer.Next,we used a database(http://starbase.sysu.edu.cn/starbase2/)to analysis GATA6-AS1 whether by m6A methylation modify regulation and predict the methyltransferases that may methylate GATA6-AS1.Furthermore,RNA immunoprecipitation experiments confirmed that GATA6-AS1 was able to bind to the m6A methylation modification enzyme.These data allowed us to clarify the ability of m6A methylase to influence the action of GATA6-AS1 and its role in the occurrence and development of gastric cancer.RESULTS Low expression levels of GATA6-AS1 were detected in gastric cancer.We also determined the effects of GATA6-AS1 overexpression on the biological function of gastric cancer cells.GATA6-AS1 had strong binding ability with the m6A demethylase FTO,which was expressed at high levels in gastric cancer and negatively correlated with the expression of GATA6-AS1.Following transfection with siRNA to knock down the expression of FTO,the expression levels of GATA6-AS1 were up-regulated.Finally,the proliferation,migration and invasion of gastric cancer cells were all inhibited following the knockdown of FTO expression.CONCLUSION During the occurrence and development of gastric cancer,the overexpression of FTO may inhibit the expression of GATA6-AS1,thus promoting the proliferation and metastasis of gastric cancer.

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.

Identification of marker genes associated with N6-methyladenosine and autophagy in ulcerative colitis

BACKGROUND Both N6-methyladenosine(m6A)methylation and autophagy are considered relevant to the pathogenesis of ulcerative colitis(UC).However,a systematic exploration of the role of the com-bination of m6A methylation and autophagy in UC remains to be performed.AIM To elucidate the autophagy-related genes of m6A with a diagnostic value for UC.METHODS The correlation between m6A-related genes and autophagy-related genes(ARGs)was analyzed.Finally,gene set enrichment analysis(GSEA)was performed on the characteristic genes.Additionally,the expression levels of four characteristic genes were verified in dextran sulfate sodium(DSS)-induced colitis in mice.RESULTS GSEA indicated that BAG3,P4HB and TP53INP2 were involved in the inflammatory response and TNF-αsignalling via nuclear factor kappa-B.Furthermore,polymerase chain reaction results showed significantly higher mRNA levels of BAG3 and P4HB and lower mRNA levels of FMR1 and TP53INP2 in the DSS group compared to the control group.CONCLUSION This study identified four m6A-ARGs that predict the occurrence of UC,thus providing a scientific reference for further studies on the pathogenesis of UC.

Recent progress in N6-methyladenosine modification in ocular surface diseases

N6-methyladenosine(m6A)modification is a reversible process promoted by“writers”,inhibited by“erasers”,and processed by“readers”.During the last decade,increasing emphasis has been placed on the underlying roles of m6A modification owing to their great importance in biological significance.The abnormal regulation of m6A modification will lead to aberrant cellular behavior and various diseases.Recently,studies have demonstrated that m6A modification is closely associated with the genesis and progression of ocular surface diseases(OSDs).This review focus on the role of m6A modification and research progress in OSDs including fungal keratitis,herpes simplex keratitis,immunerelated keratoconjunctival diseases,pterygium,ocular chemical burns,and Graves’ophthalmopathy,which may provide new insights into and prospective applications for OSDs.

Profiling of N6-methyladenosine methylation in porcine longissimus dorsi muscle and unravelling the hub gene ADIPOQ promotes adipogenesis in an m^(6)A-YTHDF1–dependent manner

Background Intramuscular fat(IMF)content is a critical indicator of pork quality,and abnormal IMF is also relevant to human disease as well as aging.Although N6-methyladenosine(m^(6)A)RNA modification was recently found to regulate adipogenesis in porcine intramuscular fat,however,the underlying molecular mechanisms was still unclear.Results In this work,we collected 20 longissimus dorsi muscle samples with high(average 3.95%)or low IMF content(average 1.22%)from a unique heterogenous swine population for m^(6)A sequencing(m^(6)A-seq).We discovered 70genes show both differential RNA expression and m^(6)A modification from high and low IMF group,including ADIPOQ and SFRP1,two hub genes inferred through gene co-expression analysis.Particularly,we observed ADIPOQ,which contains three m^(6)A modification sites within 3’untranslated and protein coding region,could promote porcine intramuscular preadipocyte differentiation in an m^(6)A-dependent manner.Furthermore,we found the YT521-B homology domain family protein 1(YTHDF1)could target and promote ADIPOQ mRNA translation.Conclusions Our study provided a comprehensive profiling of m^(6)A methylation in porcine longissimus dorsi muscle and characterized the involvement of m^(6)A epigenetic modification in the regulation of ADIPOQ mRNA on IMF deposition through an m^(6)A-YTHDF1-dependent manner.

Analysis of N6-methyladenosine-modified mRNAs in diabetic cataract

BACKGROUND Cataracts remain a prime reason for visual disturbance and blindness all over the world,despite the capacity for successful surgical replacement with artificial lenses.Diabetic cataract(DC),a metabolic complication,usually occurs at an earlier age and progresses faster than age-related cataracts.Evidence has linked N6-methyladenosine(m6A)to DC progression.However,there exists a lack of understanding regarding RNA m6A modifications and the role of m6A in DC pathogenesis.AIM To elucidate the role played by altered m6A and differentially expressed mRNAs(DEmRNAs)in DC.METHODS Anterior lens capsules were collected from the control subjects and patients with DC.M6A epitranscriptomic microarray was performed to investigate the altered m6A modifications and determine the DEmRNAs.Through Gene Ontology and pathway enrichment(Kyoto Encyclopedia of Genes and Genomes)analyses,the potential role played by dysregulated m6A modification was predicted.Real-time polymerase chain reaction was further carried out to identify the dysregulated expression of RNA methyltransferases,demethylases,and readers.RESULTS Increased m6A abundance levels were found in the total mRNA of DC samples.Bioinformatics analysis predicted that ferroptosis pathways could be associated with m6A-modified mRNAs.The levels of five methylation-related genes-RBM15,WTAP,ALKBH5,FTO,and YTHDF1-were upregulated in DC samples.Upregulation of RBM15 expression was verified in SRA01/04 cells with high-glucose medium and in samples from DC patients.CONCLUSION M6a mRNA modifications may be involved in DC progression via the ferroptosis pathway,rendering novel insights into therapeutic strategies for DC.

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.

Epigenetic N6-methyladenosine modification of RNA and DNA regulates cancer

The biological roles of N6 methylation of nucleic acids have been extensively studied.Adenine methylation of RNA is the most prevalent RNA modification and has widespread effects on RNA splicing,translation,localization,and stability.Aberrant dynamic regulation of RNA N6-methyladenosine(m6 A)has been reported in numerous human diseases,including several cancers.In recent years,eukaryotic DNA N6-methyladenosine(6 mA)has also been reported and implicated in cancer progression and tumorigenesis.In this review,we summarize the contributions of N6-methyladenosine modification to cancer biology and pathogenesis in the context of both RNA and DNA.We also highlight the clinical relevance of targeting these modifications as a therapeutic strategy for cancer.

Genome-wide map of N6-methyladenosine circular RNAs identified in mice model of severe acute pancreatitis

BACKGROUND Severe acute pancreatitis(SAP)is a deadly inflammatory disease with complex pathogenesis and lack of effective therapeutic options.N6-methyladenosine(m6A)modification of circRNAs plays important roles in physiological and pathological processes.However,the roles of m6A circRNA in the pathological process of SAP remains unknown.AIM To identify transcriptome-wide map of m6A circRNAs and to determine their biological significance and potential mechanisms in SAP.METHODS The SAP in C57BL/6 mice was induced using 4%sodium taurocholate salt.The transcriptome-wide map of m6A circRNAs was identified by m6A-modified RNA immunoprecipitation sequencing.The biological significance of circRNAs with differentially expressed m6A peaks was evaluated through gene ontology and Kyoto Encyclopedia of Genes and Genomes analysis.The underlying mechanism of m6A circRNAs in SAP was analyzed by constructing of m6A circRNAmicroRNA networks.The expression of demethylases was determined by quantitative polymerase chain reaction and western blot to deduce the possible mechanism of reversible m6A process in SAP.RESULTS Fifty-seven circRNAs with differentially expressed m6A peaks were identified by m6A-modified RNA immunoprecipitation sequencing,of which 32 were upregulated and 25 downregulated.Functional analysis of these m6A circRNAs in SAP found some important pathways involved in the pathogenesis of SAP,such as regulation of autophagy and protein digestion.In m6A circRNA–miRNA networks,several important miRNAs participated in the occurrence and progression of SAP were found to bind to these m6A circRNAs,such as miR-24-3p,miR-26a,miR-92b,miR-216b,miR-324-5p and miR-762.Notably,the total m6A level of circRNAs was reduced,while the demethylase alkylation repair homolog 5 was upregulated in SAP.CONCLUSION m6A modification of circRNAs may be involved in the pathogenesis of SAP.Our findings may provide novel insights to explore the possible pathogenetic mechanism of SAP and seek new potential therapeutic targets for SAP.

N^6-methyladenosine as a Novel Regulator of Brain Physiology and Diseases

N^6-methyladenosine(m6A)is identified as the most widespread and abundant internal chemical modification of RNA in eukaryotes.A series of proteins including methyltransferases(also known as“writers”),demethylases(also known as“erasers”),and m6A-binding proteins(also known as“readers”)were indicated to participate in the m6A methylation.m6A has emerged as a regulator of various cellular,developmental,and disease processes.Notably,there is highest abundance of m^6A methylation in brain than in other organs,which indicates that m^6A plays an essential role in brain functions.Here,we describe the general features,mechanisms,and functions of m^6A in the brain,and discuss the emerging roles of m6A in brain physiology and diseases.

Dysregulated N6-methyladenosine modification in peripheral immune cells contributes to the pathogenesis of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis(ALS)is a progressive neurogenerative disorder with uncertain origins.Emerging evidence implicates N6-methyladenosine(m6A)modification in ALS pathogenesis.Methylated RNA immunoprecipitation sequencing(MeRIP-seq)and liquid chromatography–mass spectrometry were utilized for m6A profiling in peripheral immune cells and serum proteome analysis,respectively,in patients with ALS(n=16)and controls(n=6).The single-cell transcriptomic dataset(GSE174332)of primary motor cortex was further analyzed to illuminate the biological implications of differentially methylated genes and cell communication changes.Analysis of peripheral immune cells revealed extensive RNA hypermethylation,highlighting candidate genes with differential m6A modification and expression,including C-X3-C motif chemokine receptor 1(CX3CR1).In RAW264.7 macrophages,disrupted CX3CR1 signaling affected chemotaxis,potentially influencing immune cell migration in ALS.Serum proteome analysis demonstrated the role of dysregulated immune cell migration in ALS.Cell type-specific expression variations of these genes in the central nervous system(CNS),particularly microglia,were observed.Intercellular communication between neurons and glial cells was selectively altered in ALS CNS.This integrated approach underscores m6A dysregulation in immune cells as a potential ALS contributor.

N6-methyladenosine-modified DBT alleviates lipid accumulation and inhibits tumor progression in clear cell renal cell carcinoma through the ANXA2/YAP axis-regulated Hippo pathway

Background:The mechanism of metabolism reprogramming is an unsolved problem in clear cell renal cell carcinoma(ccRCC).Recently,it was discovered that the Hippo pathway altered tumor metabolism and promoted tumor progression.Thus,this study aimed at identifying key regulators of metabolism reprogramming and the Hippo pathway in ccRCC and pinpointing potential therapeutic targets for ccRCC patients.Methods:Hippo-related gene sets and metabolic gene sets were used to screen potential regulators of the Hippo pathway in ccRCC.Public databases and samples from patients were applied to investigate the association of dihydrolipoamide branched chain transacylase E2(DBT)with ccRCC and Hippo signaling.The role of DBT was confirmed by gain or loss of function assays in vitro and in vivo.Mechanistic results were yielded by luciferase reporter assay,immunoprecipitation,mass spectroscopy,and mutational studies.Results:DBT was confirmed as a Hippo-related marker with significant prognostic predictive value,and its downregulationwas caused bymethyltransferaselike-3(METTL3)-mediated N6-methyladenosine(m6A)modification in ccRCC.Functional studies specified DBT as a tumor suppressor for inhibiting tumor progression and correcting the lipid metabolism disorder in ccRCC.Mechanistic findings revealed that annexin A2(ANXA2)interacted with the lipoyl-binding domain of DBT to activate Hippo signaling which led to decreased nuclear localization of yes1-associated transcriptional regulator(YAP)and transcriptional repression of lipogenic genes.Conclusions:This study demonstrated a tumor-suppressive role for the DBT/ANXA2/YAP axis-regulated Hippo signaling and suggested DBT as a potential target for pharmaceutical intervention in ccRCC.

N6-methyladenosine modification of CENPF mRNA facilitates gastric cancer metastasis via regulating FAK nuclear export

Background:N6-methyladenosine(m^(6)A)modification is the most common modification that occurs in eukaryotes.Although substantial effort has been made in the prevention and treatment of gastric cancer(GC)in recent years,the prognosis of GC patients remains unsatisfactory.The regulatory mechanism between m^(6)A modification and GC development needs to be elucidated.In this study,we examined m^(6)A modification and the downstream mechanism in GC.Methods:Dot blotting assays,The Cancer Genome Atlas analysis,and quantitative real‑time PCR(qRT-PCR)were used to measure the m^(6)A levels in GC tissues.Methylated RNA-immunoprecipitation sequencing and RNA sequencingwere performed to identify the targets ofm^(6)Amodification.Western blotting,Transwell,wound healing,and angiogenesis assays were conducted to examine the role of centromere protein F(CENPF)in GC in vitro.Xenograft,immunohistochemistry,and in vivo metastasis experiments were conducted to examine the role of CENPF in GC in vivo.Methylated RNA-immunoprecipitation-qPCR,RNA immunoprecipitation-qPCR and RNA pulldown assays were used to verify the m^(6)A modification sites of CENPF.Gain/loss-of-function and rescue experiments were conducted to determine the relationship between CENPF and the mitogen-activated protein kinase(MAPK)signaling pathway in GC cells.Coimmunoprecipitation,mass spectrometry,qRT-PCR,and immunofluorescence assays were performed to explore the proteins that interact with CENPF and elucidate the regulatory mechanisms between them.Results:CENPF was upregulated in GC and facilitated the metastasis of GC both in vitro and in vivo.Mechanistically,increasedm^(6)A modification of CENPF was mediated by methyltransferase 3,and this modified molecule could be recognized by heterogeneous nuclear ribonucleoprotein A2/B1(HNRNPA2B1),thereby promoting its mRNA stability.In addition,the metastatic phenotype of CENPF was dependent on the MAPK signaling pathway.Furthermore,CENPF could bind to FAK and promote its localization in the cytoplasm.Moreover,we discovered that high expression of CENPF was related to lymphatic invasion and overall survival in GC patients.Conclusions:Our findings revealed that increased m^(6)A modification of CENPF facilitates the metastasis and angiogenesis of GC through the CENPF/FAK/MAPK and epithelial-mesenchymal transition axis.CENPF expression was correlated with the clinical features of GC patients;therefore,CENPF may serve as a prognostic marker of GC.

N6-methyladenosine modified LINC00901 promotes pancreatic cancer progression through IGF2BP2/MYC axis

Accumulating evidence indicates that RNA methylation at N6-methyladenosine(m6A)plays an important regulatory role in gene expression and aberrant mRNA m6A modification is often associated with a variety of cancers.However,little is known whether and how m6A-modification impacts long non-coding RNA(lncRNA)and lncRNA-mediated tumorigenesis,particularly in pancreatic ductal adenocarcinoma(PDAC).In the present study,we report that a previously uncharacterized lncRNA,LINC00901,promotes pancreatic cancer cell growth and invasion and moreover,LINC00901 is subject to m6A modification which regulates its expression.In this regard,YTHDF1 serves as a reader for the m6A modified LINC00901 and downregulates the LINC00901 level.Notably,two conserved m6A sites in LINC00901 are critical to the recognition of LINC00901 by YTHDF1.Finally,RNA sequencing(RNA-seq)and gene function analysis revealed that LINC00901 positively regulates MYC through upregulation of IGF2BP2,a known RNA binding protein that can enhance MYC mRNA stability.Together,our results suggest that there is a LINC00901-IGF2BP2-MYC axis through which LINC00901 promotes PDAC progression in an m6A dependent manner.