Methylated RNA Immunoprecipitation Research Articles

METTL3 promotes chemoresistance in small cell lung cancer by inducing mitophagy

BackgroundSmall cell lung cancer (SCLC) is the most aggressive subtype of lung cancer. Although most patients are initially sensitive to first-line combination chemotherapy with cisplatin and etoposide, chemotherapy drug resistance easily develops and quickly leads to tumour progression. Therefore, understanding the mechanisms of chemotherapy drug resistance and how to reverse it is key to improving the prognosis of patients with SCLC. Moreover, N6-methyladenosine (m6A) is the most abundant mRNA modification and is catalysed by the methyltransferase complex, in which methyltransferase-like 3 (METTL3) is the sole catalytic subunit.MethodsThe effects of METTL3 on chemoresistance in SCLC cells were determined using qRT–PCR, Western blotting, immunohistochemistry, cell counting kit (CCK-8) assays, flow cytometry, and tumorigenicity experiments. Methylated RNA immunoprecipitation sequencing (MeRIP-seq), MeRIP qPCR, immunofluorescence, and drug inhibitor experiments were performed to confirm the molecular mechanism of Decapping Protein 2 (DCP2), which is involved in the chemoresistance of SCLC.ResultsIn the present study, we found that METTL3 is a marker for poor SCLC prognosis, and it is highly expressed in chemoresistant SCLC cells. METTL3 promotes SCLC chemoresistance by positively regulating mitophagy. METTL3 induces m6A methylation of DCP2 and causes the degradation of DCP2, which promotes mitochondrial autophagy through the Pink1-Parkin pathway, leading to chemotherapy resistance. We also found that STM2457, a novel METTL3 inhibitor, can reverse SCLC chemoresistance.ConclusionsThe m6A methyltransferase METTL3 regulates Pink1-Parkin pathway-mediated mitophagy and mitochondrial damage in SCLC cells by targeting DCP2, thereby promoting chemotherapy resistance in patients with SCLC.

Epigenetic combined with transcriptomic analysis of the m6A methylome after spared nerve injury-induced neuropathic pain in mice.

Epigenetic changes in the spinal cord play a key role in the initiation and maintenance of nerve injury-induced neuropathic pain. N6-methyladenosine (m6A) is one of the most abundant internal RNA modifications and plays an essential function in gene regulation in many diseases. However, the global m6A modification status of mRNA in the spinal cord at different stages after neuropathic pain is unknown. In this study, we established a neuropathic pain model in mice by preserving the complete sural nerve and only damaging the common peroneal nerve. High-throughput methylated RNA immunoprecipitation sequencing results showed that after spared nerve injury, there were 55 m6A methylated and differentially expressed genes in the spinal cord. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway results showed that m6A modification triggered inflammatory responses and apoptotic processes in the early stages after spared nerve injury. Over time, the differential gene function at postoperative day 7 was enriched in "positive regulation of neurogenesis" and "positive regulation of neural precursor cell proliferation." These functions suggested that altered synaptic morphological plasticity was a turning point in neuropathic pain formation and maintenance. Results at postoperative day 14 suggested that the persistence of neuropathic pain might be from lipid metabolic processes, such as "very-low-density lipoprotein particle clearance," "negative regulation of cholesterol transport" and "membrane lipid catabolic process." We detected the expression of m6A enzymes and found elevated mRNA expression of Ythdf2 and Ythdf3 after spared nerve injury modeling. We speculate that m6A reader enzymes also have an important role in neuropathic pain. These results provide a global landscape of mRNA m6A modifications in the spinal cord in the spared nerve injury model at different stages after injury.

ALKBH5-induced circular RNA NRIP1 promotes glycolysis in thyroid cancer cells by targeting PKM2.

Although circular RNAs (circRNAs) are involved in cell proliferation, differentiation, apoptosis, and invasion, the underlying regulatory mechanisms of circRNAs in thyroid cancer have not been fully elucidated. This article aimed to study the role of circRNA regulated by N6-methyladenosine modification in papillary thyroid cancer (PTC). Quantitative real-time PCR, western blotting, and immunohistochemistry were used to investigate the expressions of circRNA nuclear receptor-interacting protein 1 (circNRIP1) in PTC tissues and adjacent noncancerous thyroid tissues. In vitro and in vivo assays were carried out to assess the effects of circNRIP1 on PTC glycolysis and growth. The N6-methyladenosine mechanisms of circNRIP1 were evaluated by methylated RNA immunoprecipitation sequencing, luciferase reporter gene, and RNA stability assays. Results showed that circNRIP1 levels were significantly upregulated in PTC tissues. Furthermore, elevated circNRIP1 levels in PTC patients were correlated with high tumor lymph node metastasis stage and larger tumor sizes. Functionally, circNRIP1 significantly promoted glycolysis, PTC cell proliferation in vitro, and tumorigenesis in vivo. Mechanistically, circNRIP1 acted as a sponge for microRNA (miR)-541-5p and miR-3064-5p and jointly upregulated pyruvate kinase M2 (PKM2) expression. Knockdown of m6 A demethylase α-ketoglutarate-dependent dioxygenase alkB homolog 5 (ALKBH5) significantly enhanced circNRIP1 m6 A modification and upregulated its expression. These results show that ALKBH5 knockdown upregulates circNRIP1, thus promoting glycolysis in PTC cells. Therefore, circNRIP1 can be a prognostic biomarker and therapeutic target for PTC by acting as a sponge for oncogenic miR-541-5p and miR-3064-5p to upregulate PKM2 expression.

Alkylation repair homolog 3-regulated esophageal squamous cell carcinoma associated long non-coding RNA 1 is required for maintaining the stemness of esophageal cancer

N1-methyladenosine (m1A) RNA modification represents one of the essential post-transcriptional modifications in gene expression regulation. Long non-coding RNAs (lncRNAs) are involved in the development of malignant tumors, including esophageal cancer (ESCA). However, whether m1A can regulate that lncRNA in cancer cells remains unclear. ESCA cell lines TE1 and KYSE70 were used for functional experiments. The mRNA and protein levels were detected by quantitative reverse transcription polymerase chain reaction and Western blot, respectively. Colony formation and tumor sphere formation assays were used for evaluating ESCA stemness. The m1A modification on esophageal squamous cell carcinoma associated long non-coding RNA 1 (ESCCAL-1) transcript was examined by methylated RNA immunoprecipitation. In this study, we report that RNA m1A demethylase alkylation repair homolog 3 (ALKBH3)-mediated ESCCAL-1 is implicated in maintaining stem cell-like properties of ESCA. Clinically, ESCCAL-1 was up-regulated in ESCA and positively correlated with tumor stage. In addition, patients with higher ESCCAL-1 expression in tumors had shorter median survival. Functionally, the knockdown of ESCCAL-1 attenuated the stemness of ESCA cells as indicated by decreased sphere formation and colony formation capacities, while overexpression of ESCCAL-1 elicits the opposite biological effects. Moreover, ESCCAL-1 manipulation positively regulated both mRNA and protein levels of KLF4 and CD44, two stemness-related markers. Mechanistically, ALKBH3 upregulated ESCCAL-1 expression in an m1A demethylation-dependent manner. Notably, the downregulation of ALKBH3 mimicked the effects of ESCCAL-1 deficiency on ESCA stemness, and this phenomenon is significantly reversed by the enforced expression of ESCCAL-1. Our results revealed the role of m1A-mediated ESCCAL-1 in ESCA self-renewal, which expands the understanding of lncRNA post-transcriptional modification in cancer development.

M6A methyltransferase WTAP regulates myocardial ischemia reperfusion injury through YTHDF1/FOXO3a signaling.

N6-methyladenosine (m6A) is emerging as an essential regulator in the progression of myocardial ischemia reperfusion (I/R) injury. However, the in-depth functions and mechanisms for m6A are still unclear. This work aimed to explore the potential functions and mechanisms for myocardial I/R injury. In this study, m6A methyltransferase WTAP and m6A modification level elevated in the hypoxia/reoxygenation (H/R) induced rat cardiomyocytes (H9C2) and I/R injury rat model. Bio-functional cellular experiments demonstrated that knockdown of WTAP remarkably released the proliferation and reduced the apoptosis and inflammatory cytokines induced by H/R. Moreover, exercise training alleviated WTAP level in exercise-trained rats. Mechanistically, methylated RNA immunoprecipitation sequencing (MeRIP-Seq) revealed that a remarkable m6A modification site was found in the 3'-UTR of FOXO3a mRNA. Moreover, WTAP triggered the installation of m6A modification on FOXO3a mRNA through m6A reader YTHDF1, thereby enhancing the stability of FOXO3a mRNA. Collectively, WTAP/YTHDF1/m6A/FOXO3a axis regulates the myocardial I/R injury progression, which provides new insights for the treatment of myocardial injury.

The Important Role of m6A-Modified circRNAs in the Differentiation of Intramuscular Adipocytes in Goats Based on MeRIP Sequencing Analysis.

Intramuscular fat contributes to the improvement of goat meat quality. N6-Methyladenosine (m6A)-modified circular RNAs play important roles in adipocyte differentiation and metabolism. However, the mechanisms by which m6A modifies circRNA before and after differentiation of goat intramuscular adipocytes remain poorly understood. Here, we performed methylated RNA immunoprecipitation sequencing (MeRIP-seq) and circRNA sequencing (circRNA-seq) to determine the distinctions in m6A-methylated circRNAs during goat adipocyte differentiation. The profile of m6A-circRNA showed a total of 427 m6A peaks within 403 circRNAs in the intramuscular preadipocytes group, and 428 peaks within 401 circRNAs in the mature adipocytes group. Compared with the intramuscular preadipocytes group, 75 peaks within 75 circRNAs were significantly different in the mature adipocytes group. Furthermore, the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses of intramuscular preadipocytes and mature adipocytes showed that the differentially m6A-modified circRNAs were enriched in the PKG signaling pathway, endocrine and other factor-regulated calcium reabsorption, lysine degradation, etc. m6A-circRNA-miRNA-mRNA interaction networks predicted the potential m6A-circRNA regulation mechanism in different goat adipocytes. Our results indicate that there is a complicated regulatory relationship between the 12 upregulated and 7 downregulated m6A-circRNAs through 14 and 11 miRNA mediated pathways, respectively. In addition, co-analysis revealed a positive association between m6A abundance and levels of circRNA expression, such as expression levels of circRNA_0873 and circRNA_1161, which showed that m6A may play a vital role in modulating circRNA expression during goat adipocyte differentiation. These results would provide novel information for elucidating the biological functions and regulatory characteristics of m6A-circRNAs in intramuscular adipocyte differentiation and could be helpful for further molecular breeding to improve meat quality in goats.

RNA methylation in plants: An overview.

RNA methylation is an important post-transcriptional modification that influences gene regulation. Over 200 different types of RNA modifications have been identified in plants. In animals, the mystery of RNA methylation has been revealed, and its biological role and applications have become increasingly clear. However, RNA methylation in plants is still poorly understood. Recently, plant science research on RNA methylation has advanced rapidly, and it has become clear that RNA methylation plays a critical role in plant development. This review summarizes current knowledge on RNA methylation in plant development. Plant writers, erasers, and readers are highlighted, as well as the occurrence, methods, and software development in RNA methylation is summarized. The most common and abundant RNA methylation in plants is N6-methyladenosine (m6A). In Arabidopsis, mutations in writers, erasers, and RNA methylation readers have affected the plant's phenotype. It has also been demonstrated that methylated TRANSLATIONALLY CONTROLLED TUMOR PROTEIN 1-messenger RNA moves from shoot to root while unmethylated TCTP1-mRNA does not. Methylated RNA immunoprecipitation, in conjunction with next-generation sequencing, has been a watershed moment in plant RNA methylation research. This method has been used successfully in rice, Arabidopsis, Brassica, and maize to study transcriptome-wide RNA methylation. Various software or tools have been used to detect methylated RNAs at the whole transcriptome level; the majority are model-based analysis tools (for example, MACS2). Finally, the limitations and future prospects of methylation of RNA research have been documented.

The Suppression Effects of Fat Mass and Obesity Associated Gene on the Hair Follicle-Derived Neural Crest Stem Cells Differentiating into Melanocyte by N6-Methyladenosine Modifying Microphthalmia-Associated Transcription Factor.

Melanocyte (MC), derived from neural crest stem cell (NCSC), are involved in the production of melanin. The mechanism by which NCSC differentiates to MC remains unclear. N6-methyladenosine (m6A) modification was applied to discuss the potential mechanism. NCSCs were isolated from hair follicles of rats, and were obtained for differentiation. Cell viability, tyrosinase secretion and activity, and transcription factors were combined to evaluated the MC differentiation. RT-qPCR was applied to determine mRNA levels, and western blot were used for protein expression detection. Total m6A level was measured using methylated RNA immunoprecipitation (MeRIP) assay, and RNA immunoprecipitation was used to access the protein binding relationship. In current work, NCSCs were successfully differentiated into MCs. Fat mass and obesity associated gene (FTO) was aberrant downregulated in MCs, and elevated FTO suppressed the differentiation progress of NCSCs into MCs. Furthermore, microphthalmia-associated transcription factor (Mitf), a key gene involved in MC synthesis, was enriched by FTO in a m6A modification manner and degraded by FTO. Meanwhile, the suppression functions of FTO in the differentiation of NCSCs into MCs were reversed by elevated Mitf. In short, FTO suppressed the differentiating ability of hair follicle-derived NCSCs into MCs by m6A modifying Mitf.

WTAP regulates autophagy in colon cancer cells by inhibiting FLNA through N6-methyladenosine

ABSTRACT Our study investigated the role of WTAP in colon cancer. We employed experiments including m6A dot blot hybridization, methylated RNA immunoprecipitation, dual-luciferase, and RNA immunoprecipitation to investigate the regulatory mechanism of WTAP. Western blot was performed to analyze the expression of WTAP, FLNA and autophagy-related proteins in cells. Our results confirmed the up-regulation of WTAP in colon cancer and its promoting effect on proliferation and inhibiting effect on apoptosis. FLNA was the downstream gene of WTAP and WTAP-regulated m6A modification led to post-transcriptional repression of FLNA. The rescue experiments showed that WTAP/FLNA could inhibit autophagy. WTAP-mediated m6A modification was confirmed to be crucial in colon cancer development, providing new insights into colon cancer therapy.

Transcriptome-wide analysis of mRNA N6 -methyladenosine modification in the embryonic development of Spodoptera frugiperda.

N6 -methyladenosine (m6 A) RNA is the most abundant modification of mRNA, and has been demonstrated in regulating various post-transcriptional processes. Many studies have shown that m6 A methylation plays key roles in sex determination, neuronal functions, and embryonic development in Drosophila and mammals. Here, we analyzed transcriptome-wide profile of m6 A modification in the embryonic development of the destructive agricultural pest Spodoptera frugiperda. We found that the 2 key mRNA m6 A methyltransferases SfrMETTL3 and SfrMETTL14 have high homologies with other insects and mammals, suggesting that SfrMETTL3 and SfrMETTL14 may have conserved function among different species. From methylated RNA immunoprecipitation sequencing analysis, we obtained 46869 m6 A peaks representing 8587 transcripts in the 2-h embryos after oviposition, and 41389 m6 A peaks representing 9230 transcripts in the 24-h embryos. In addition, 5995 m6 A peaks were differentially expressed including 3752 upregulated and 2243 downregulated peaks. Functional analysis with Gene Ontology and Kyoto Encyclopedia of Genes and Genomes suggested that differentially expressed m6 A peak-modified genes were enriched in cell and organ development between the 2- and 24-h embryos. By conjoint analysis of methylated RNA immunoprecipitation-seq and RNA-seq data, we found that RNA m6 A methylation may regulate the transcriptional levels of genes related to tissue and organ development from 2- to 24-h embryos. Our study reveals the role of RNA m6 A epigenetic regulation in the embryonic development of S. frugiperda, and provides new insights for the embryonic development of insects.

Conserved reduction of m6A RNA modifications during aging and neurodegeneration is linked to changes in synaptic transcripts

N6-methyladenosine (m6A) regulates mRNA metabolism. While it has been implicated in the development of the mammalian brain and in cognition, the role of m6A in synaptic plasticity, especially during cognitive decline, is not fully understood. In this study, we employed methylated RNA immunoprecipitation sequencing to obtain the m6A epitranscriptome of the hippocampal subregions CA1, CA3, and the dentate gyrus and the anterior cingulate cortex (ACC) in young and aged mice. We observed a decrease in m6A levels in aged animals. Comparative analysis of cingulate cortex (CC) brain tissue from cognitively intact human subjects and Alzheimer's disease (AD) patients showed decreased m6A RNA methylation in AD patients. m6A changes common to brains of aged mice and AD patients were found in transcripts linked to synaptic function including calcium/calmodulin-dependent protein kinase 2 (CAMKII) and AMPA-selective glutamate receptor 1 (Glua1). We used proximity ligation assays to show that reduced m6A levels result in decreased synaptic protein synthesis as exemplified by CAMKII and GLUA1. Moreover, reduced m6A levels impaired synaptic function. Our results suggest that m6A RNA methylation controls synaptic protein synthesis and may play a role in cognitive decline associated with aging and AD.

The importance of m6A topology in chicken embryo mRNA: a precise mapping of m6A at the conserved chicken β-actin zipcode.

N6-methyladenosine (m6A) in mRNA regulates almost every stage in the mRNA life cycle, and the development of methodologies for the high-throughput detection of methylated sites in mRNA using m6A-specific methylated RNA immunoprecipitation with next-generation sequencing (MeRIPSeq) or m6A individual-nucleotide-resolution cross-linking and immunoprecipitation (miCLIP) have revolutionized the m6A research field. Both of these methods are based on immunoprecipitation of fragmented mRNA. However, it is well documented that antibodies often have nonspecific activities, thus verification of identified m6A sites using an antibody-independent method would be highly desirable. We mapped and quantified the m6A site in the chicken β-actin zipcode based on the data from chicken embryo MeRIPSeq results and our RNA-Epimodification Detection and Base-Recognition (RedBaron) antibody-independent assay. We also demonstrated that methylation of this site in the β-actin zipcode enhances ZBP1 binding in vitro, while methylation of a nearby adenosine abolishes binding. This suggests that m6A may play a role in regulating localized translation of β-actin mRNA, and the ability of m6A to enhance or inhibit a reader protein's RNA binding highlights the importance of m6A detection at nucleotide resolution.

RBM15 suppresses hepatic insulin sensitivity of offspring of gestational diabetes mellitus mice via m6A-mediated regulation of CLDN4

BackgroundGestational diabetes Mellitus (GDM) is a common pregnancy-specific disease with high morbidity, which is linked to a high risk of obesity and diabetes in offspring. N6-methyladenosine modification of RNA is emerging as an important epigenetic mechanism that is widely manifested in many diseases. This study aimed to investigate the mechanism of m6A methylation in metabolic syndrome in offspring result from intrauterine hyperglycemia.MethodsGDM mice were established by feeding a high-fat diet 1 weeks before pregnancy. The m6A RNA methylation quantification kit was used to detect liver tissue methylation levels. PCR array was used to determine the expression of the m6A methylation modification enzyme. Immunohistochemistry, qRT-PCR, and western blot were used to examine the expression of RBM15, METTL13, IGF2BP1, and IGF2BP2. Subsequently, methylated RNA immunoprecipitation sequencing combined with mRNA sequencing, followed by dot blot and glucose uptake tests, were performed.ResultsIn this study, we found that offspring from a GDM mother were more vulnerable to glucose intolerance and insulin resistance. GC–MS revealed significant metabolic changes including saturated fatty acids and unsaturated fatty acids in liver of GDM offspring. We also demonstrated that global mRNA m6A methylation level was significantly increased in the fetal liver of GDM mice, indicating epigenetic change may have a strong relationship with the mechanism of metabolism syndrome. Concordantly, RBM15, the RNA binding methyltransferase, was upregulated in the liver. In vitro, RBM15 suppressed insulin sensitivity and increased insulin resistance through m6A-regulated epigenetic inhabitation of CLDN4. Moreover, MeRIP-sequencing and mRNA-sequencing revealed that differently regulated genes with differential m6A peaks were enriched in metabolic pathways.ConclusionOur study revealed the essential role of RBM15 in insulin resistance and the effect of RBM15-regulated m6A modification in the metabolic syndrome of offspring of GDM mice.

Transcriptome-wide assessment of N6-methyladenosine modification identifies different gene expression and infection-associated pathways in Treponema pallidum-infected macrophage

BackgroundTreponema pallidum (Tp) is a widespread and destructive pathogen that leads to syphilis. As the acknowledged executor of host immunity, macrophage plays vital roles in combating the invasion and migration of Tp. However, the mechanisms of these processes are largely unknown, especially the critical driver genes and associated modifications. ObjectiveWe aimed to systematically dissect the global N6-methyladenosine (m6A) RNA modification patterns in Tp-infected macrophages. MethodsThe RNA of Tp-infected/non-infected macrophage was extracted, followed by mRNA sequencing and methylated RNA immunoprecipitation (MeRIP) sequencing. Bioinformatics analysis was executed by m6A peaks and motifs identification, Gene ontology and signaling pathways analysis of differentially expressed genes, and comprehensive comparison. The m6A levels were measured by RNA Methylation Assay, and m6A modified genes were determined by qPCR. ResultsTotally, 2623 unique and 3509 common m6A peaks were proved along with related transcripts in Tp-infected macrophages. The common m6A-related genes were enriched in the signals of oxidative stress, cell differentiation, and angiogenesis, while unique genes in those of metabolism, inflammation, and infection. And differentially expressed transcripts revealed various biological processes and pathways associated with catabolic and infection. They also experienced comprehensive analysis due to hyper-/hypo-methylation. And the m6A level of macrophage was elevated, along with qPCR validation of specific genes. ConclusionWith a particular m6A transcriptome-wide map, our study provides unprecedented insights into the RNA modification of macrophage stimulated by Tp in vitro, which partially differs from other infections and may provide clues to explore the immune process for syphilis.

Splice site m6A methylation prevents binding of DGCR8 to suppress KRT4 pre-mRNA splicing in oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is the 11th most prevalent tumor worldwide. Despite advantages of therapeutic approaches, the 5-year survival rate of patients with OSCC is less than 50%. It is urgent to elucidate mechanisms underlying OSCC progression for developing novel treatment strategies. Our recent study has revealed that Keratin 4 (KRT4) suppresses OSCC development, which is downregulated in OSCC. Nevertheless, the mechanism downregulating KRT4 in OSCC remains unknown. In this study, touchdown PCR was utilized to detect KRT4 pre-mRNA splicing, while m6A RNA methylation was identified by methylated RNA immunoprecipitation (MeRIP). Besides, RNA immunoprecipitation (RIP) was used to determine RNA-protein interaction. Herein, this study indicated that intron splicing of KRT4 pre-mRNA was suppressed in OSCC. Mechanistically, m6A methylation of exon-intron boundaries prevented intron splicing of KRT4 pre-mRNA in OSCC. Besides, m6A methylation suppressed the binding of splice factor DGCR8 microprocessor complex subunit (DGCR8) to exon-intron boundaries in KRT4 pre-mRNA to prohibit intron splicing of KRT4 pre-mRNA in OSCC. These findings revealed the mechanism downregulating KRT4 in OSCC and provided potential therapeutic targets for OSCC.

N6-methyladenosine RNA is modified in the rat hippocampus following traumatic brain injury with hypothermia treatment.

Recent studies have suggested a role for N6-methyladenosine (m6A) modification in neurological diseases. Hypothermia, a commonly used treatment for traumatic brain injury, plays a neuroprotective role by altering m6A modifications. In this study, methylated RNA immunoprecipitation sequencing (MeRIP-Seq) was applied to conduct a genome-wide analysis of RNA m6A methylation in the rat hippocampus of Sham and traumatic brain injury (TBI) groups. In addition, we identified the expression of mRNA in the rat hippocampus after TBI with hypothermia treatment. Compared with the Sham group, the sequencing results of the TBI group showed that 951 different m6A peaks and 1226 differentially expressed mRNAs were found. We performed cross-linking analysis of the data of the two groups. The result showed that 92 hyper-methylated genes were upregulated, 13 hyper-methylated genes were downregulated, 25 hypo-methylated genes were upregulated, and 10 hypo-methylated genes were downregulated. Moreover, a total of 758 differential peaks were identified between TBI and hypothermia treatment groups. Among these differential peaks, 173 peaks were altered by TBI and reversed by hypothermia treatment, including Plat, Pdcd5, Rnd3, Sirt1, Plaur, Runx1, Ccr1, Marveld1, Lmnb2, and Chd7. We found that hypothermia treatment transformed some aspects of the TBI-induced m6A methylation landscape of the rat hippocampus.

M6A-modified circRNA MYO1C participates in the tumor immune surveillance of pancreatic ductal adenocarcinoma through m6A/PD-L1 manner

Emerging evidence indicates the critical roles of N6-methyladenosine (m6A) modification in human cancers. Herein, our work reported that a novel m6A-modified circRNA from the MYO1C gene, circMYO1C, upregulated in the pancreatic ductal adenocarcinoma (PDAC). Our findings demonstrated that circMYO1C is highly expressed in PDAC tissues. Functionally, circMYO1C promoted the proliferation and migration of PDAC cells in vitro and its silencing reduced the tumor growth in vivo. Mechanistically, circMYO1C cyclization was mediated by m6A methyltransferase METTL3. Moreover, methylated RNA immunoprecipitation sequencing (MeRIP-seq) unveiled the remarkable m6A modification on PD-L1 mRNA. Moreover, circMYO1C targeted the m6A site of PD-L1 mRNA to enhance its stability by cooperating with IGF2BP2, thereby accelerating PDAC immune escape. In conclusion, these findings highlight the oncogenic role of METTL3-induced circMYO1C in PDAC tumorigenesis via an m6A-dependent manner, inspiring a novel strategy to explore PDAC epigenetic therapy.

YTHDC1 maintains trophoblasts function by promoting degradation of m6A-modified circMPP1

N6-methyladenosine (m6A) is the most abundant mRNA internal modification in eukaryotic mRNAs. This study focuses on the effect of circMPP1 on placental villi function and the molecular mechanism. First, differentially expressed circular RNAs (circRNAs) in placenta tissues of large-for-gestational-age(LGA) neonates were screened by m6A-circRNA Epitranscriptomic Microarray and bioinformatics analyses. The abnormal expression of circMPP1 in placental tissues and cell lines was validated by RT-qPCR. In-vitro and in-vivo functional experiments were performed to evaluate the role of circMPP1 in placental impairment and fetal dysplasia. The interacting proteins of circMPP1 were identified and validated using RNA pull-down, RNA immunoprecipitation, fluorescence in situ hybridization, and immunofluorescence experiments. Protein interactions and expression levels were detected by Co-immunoprecipitation and western blot analysis. The m6A modification in circMPP1 was verified by methylated RNA immunoprecipitation assay. Bioinformatics analyses showed that circMPP1 was highly expressed in tissues with disordered placental function. In-vitro and in-vivo functional experiments showed that circMPP1 inhibited the function of placental villi. Further mechanism analyses showed that circMPP1 activated the NF-kappa B and MAPK3 signaling pathways. In addition, the m6A “reader” protein YTHDC1 was found to reduce circMPP1 expression via m6A modification. In conclusion, this study demonstrates that YTHDC1 maintains trophoblasts function by promoting degradation of m6A-mediated circMPP1.

Methyltransferase-like 3 aggravates endoplasmic reticulum stress in preeclampsia by targeting TMBIM6 in YTHDF2-dependent manner

BackgroundWith the increasing morbidity and mortality of preeclampsia (PE), it has posed a huge challenge to public health. Previous studies have reported endoplasmic reticulum (ER) stress could contribute to trophoblastic dysfunction which was associated with the N6-methyladenosine (m6A) modification by methyltransferase-like 3 (METTL3), resulting in PE. However, little was known about the relationship between METTL3 and ER stress in PE. Thus, in vitro and in vivo studies were performed to clarify the mechanism about how METTL3 affects the trophoblasts under ER stress in PE and to explore a therapeutic approach for PE.MethodsAn ER stress model in HTR-8/SVneo cells and a preeclamptic rat model were used to study the mechanism and explore a therapeutic approach for PE. Western blot, immunohistochemistry, quantitative reverse transcription-polymerase chain reaction (qRT-PCR), and methylated RNA immunoprecipitation (MeRIP)-qPCR were performed to detect the protein, RNA, and methylated transmembrane BAX inhibitor motif containing 6 (TMBIM6) expression levels. The m6A colorimetric and mRNA stability assays were used to measure the m6A levels and TMBIM6 stability, respectively. Short hairpin RNAs (shRNAs) were used to knockdown METTL3 and YTH N6-methyladenosine RNA binding protein 2 (YTHDF2). Flow cytometry and Transwell assays were performed to evaluate the apoptosis and invasion abilities of trophoblasts.ResultsUpregulated METTL3 and m6A levels and downregulated TMBIM6 levels were observed in preeclamptic placentas under ER stress. The ER stress model was successfully constructed, and knockdown of METTL3 had a beneficial effect on HTR-8/SVneo cells under ER stress as it decreased the levels of methylated TMBIM6 mRNA. Moreover, overexpression of TMBIM6 was beneficial to HTR-8/SVneo cells under ER stress as it could neutralize the harmful effects of METTL3 overexpression. Similar to the knockdown of METTL3, downregulation of YTHDF2 expression resulted in the increased expression and mRNA stability of TMBIM6. Finally, improved systemic symptoms as well as protected placentas and fetuses were demonstrated in vivo.ConclusionsMETTL3/YTHDF2/TMBIM6 axis exerts a significant role in trophoblast dysfunction resulting in PE while inhibiting METTL3 may provide a novel therapeutic approach for PE.

Prognostic and immunological role of sulfatide-related lncRNAs in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the most common primary malignancy of the liver. Long non-coding RNAs (lncRNAs) play important roles in the occurrence and development of HCC through multiple pathways. Our previous study reported the specific molecular mechanism for sulfatide regulation of integrin αV expression and cell adhesion in HCC cells through lncRNA AY927503. Next, it is necessary to identify more sulfatide-related lncRNAs, explore their clinical signifcance, and determine new targeted treatment strategies. Microarrays were used to screen a complete set of lncRNAs with different expression profiles in sulfatide-treated cells. Sulfatide-related lncRNAs expression data and corresponding HCC patient survival information were obtained from the The Cancer Genome Atlas (TCGA) database, and the prognosis prediction model was constructed based on Cox regression analysis. Methylated RNA immunoprecipitation with next generation sequencing (MeRIP-seq) was used to detemine the effect of sulfatide on lncRNAs m6A modification. Tumor Immune Estimation Resource (TIMER) and Gene set nnrichment analysis (GSEA) were utilized to enrich the immune and functional pathways of sulfatide-related lncRNAs. A total of 85 differentially expressed lncRNAs (|Fold Change (FC)|>2, P<0.05) were screened in sulfatide-treated HCC cells. As a result, 24 sulfatide-related lncRNAs were highly expressed in HCC tissues, six of which were associated with poor prognosis in HCC patients. Based on thses data, a sulfatide-related lncRNAs prognosis assessment model for HCC was constructed. According to this risk score analysis, the overall survival (OS) curve showed that the OS of high-risk patients was significantly lower than that of low-risk patients (P<0.05). Notably, the expression difference in sulfatide-related lncRNA NRSN2-AS1 may be related to sulfatide-induced RNA m6A methylation. In addition, the expression level of NRSN2-AS1 was significantly positively correlated with immune cell infiltration in HCC and participated in the peroxisome and Peroxisome proliferator-activated receptor (PPAR) signaling pathways. In conclusion, sulfatide-related lncRNAs might be promising prognostic and therapeutic targets for HCC.