Methylated RNA Immunoprecipitation Research Articles

ALKBH5-mediated m6 A demethylation of TIRAP mRNA promotes radiation-induced liver fibrosis and decreases radiosensitivity of hepatocellular carcinoma.

Radiation-induced hepatic stellate cell (HSC) activation promotes radiation-induced liver fibrosis (RILF), a complication for hepatocellular carcinoma (HCC) radiotherapy. The demethylase alpha-ketoglutarate-dependent dioxygenase alkB homolog 5 (ALKBH5) decreases N6-methyladenylate methylation (m6 A) modification of RNA, while its role in regulating RILF pathogenesis and HCC radiosensitivity remains unknown. Methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA-sequencing (RNA-seq) were used to screen target genes regulated by ALKBH5. HSC with altered ALKBH5 expression was used to assess irradiation-induced HSC activation and the effect of HSC on recruitment and polarisation of monocytes. Key cytokines in medium from irradiated HSC-educated monocytes were identified by cytokine array detection. The effects of blocking ALKBH5 and key cytokines on RILF and HCC radiosensitivity were also evaluated. Radiation-induced ALKBH5 expression in HSC mediated m6 A demethylation of toll-interleukin 1 receptor domain containing adaptor protein (TIRAP) mRNA and activated its downstream NF-κB and JNK/Smad2 pathways to promote HSC activation. Additionally, ALKBH5 regulated CCL5 secretion by irradiated HSC to promote monocyte recruitment and M2 macrophage polarisation. Notably, polarised monocytes secreted CCL20 to up-regulate ALKBH5 expression in HSC, and reduce HCC radiosensitivity by activating ALKBH5/TIRAP axis in HCC cells. ALKBH5 knockdown-combined CCR6 (CCL20 receptor) inhibitor significantly alleviated RILF and improved HCC radiosensitivity in mice. HCC patients with high ALKBH5 and TIRAP expression were prone to radiation-induced liver injury and poor tumour response to radiotherapy. Collectively, irradiation up-regulates ALKBH5 in HSC to mediate monocyte recruitment and M2 polarisation and form positive feedback to promote RILF and reduce HCC radiosensitivity. The dual roles of ALKBH5 as a microenvironmental regulator and radiosensitisation target provide new ideas for RILF prevention and radiosensitisation of HCC.

Targeting m6A reader YTHDF1 augments antitumour immunity and boosts anti-PD-1 efficacy in colorectal cancer

ObjectiveThe role of N6-methyladenosine (m6A) in tumour immune microenvironment (TIME) remains understudied. Here, we elucidate function and mechanism of YTH N6-methyladenosine RNA binding protein 1 (YTHDF1) in colorectal cancer (CRC)...

The potential role of N6-methyladenosine modification of LncRNAs in contributing to the pathogenesis of chronic glomerulonephritis.

Increasing evidence indicates that N6-methyladenosine (m6A) modification of mRNAs has been shown to play a critical role in the occurrence and development of many diseases, while little is known about m6A modification in long non-coding RNAs (LncRNAs). Our study aims to investigate the potential functions of LncRNA m6A modifications in lipopolysaccharide (LPS)-induced mouse mesangial cells (MMCs), providing us with a new perspective on the molecular mechanisms of chronic glomerulonephritis (CGN) pathogenesis. Differentially methylated LncRNAs were identified by Methylated RNA immunoprecipitation sequencing (MeRIP-seq). LncRNA-mRNA and LncRNA-associated LncRNA-miRNA-mRNA (CeRNA) networks were constructed by bioinformatics analysis. Furthermore, we utilized gene ontology (GO) and pathway enrichment analyses (KEGG) to explore target genes from co-expression networks. In addition, the total level of m6A RNA methylation and expression of methyltransferase and pro-inflammatory cytokines were detected by the colorimetric quantification method and western blot, respectively. Cell viability and cell cycle stage were detected by cell counting kit-8 (CCK-8) and flow cytometry. In total, 1141 differentially m6A-methylated LncRNAs, including 529 hypermethylated LncRNAs and 612 hypomethylated LncRNAs, were determined by MeRIP-seq. The results of GO and KEGG analysis revealed that the target mRNAs were mainly enriched in signal pathways, such as the NF-kappa B signaling pathway, MAPK signaling pathway, Toll-like receptor signaling pathway, and apoptosis signaling pathway. In addition, higher METTL3 expression was found in CGN kidney tissues using the GEO database. METTL3 knockdown in MMC cells drastically reduced the levels of m6A RNA methylation, pro-inflammatory cytokines IL6 and TNF-α, and inhibited cell proliferation and cycle progression. Our findings provide a basis and novel insight for further investigations of m6A modifications in LncRNAs for the pathogenesis of CGN.

Differential methylation of circRNA m6A in an APP/PS1 Alzheimer's disease mouse model.

Alzheimer's disease (AD) is a chronic neurological disease characterized by memory loss and progressive cognitive impairment. The characteristic AD pathologies include extracellular senile plaques formed by β‑amyloid protein deposition, neurofibrillary tangles formed by hyper‑phosphorylation of τ protein and neuronal loss caused by glial cell proliferation. However, the pathogenesis of AD is still unclear. Dysregulation of RNA methylation is associated with biological processes, including neurodevelopment and neurodegenerative disease. N6‑methyladenosine (m6A) is the main modification in eukaryotic RNA and may be associated with the pathophysiology of AD. Circular RNA (circRNA) is a new type of evolutionarily conserved non‑coding RNA without 5'‑cap and 3'‑polyadenylic acid tail. circRNA undergoes m6A RNA methylation and may be involved in the pathogenesis of AD. In the present study, high‑throughput sequencing was performed to assess the degree of circRNA m6A methylation in APP/PS1 AD and C57BL/6 mice. These results suggested that circRNA m6A methylation in AD mice was markedly altered compared to the control group. Furthermore, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis was used to predict associated pathways; genes with different circRNA m6A methylation in AD mice were associated with 'axon guidance', 'long‑term potentiation', 'glutamatergic synapse', 'cholinergic synapse', 'GABAergic synapse' and 'long‑term depression'. Methylated RNA immunoprecipitation reverse transcription‑quantitative PCR demonstrated that among the eight selected circRNA m6A genes, there were five genes that demonstrated significantly increased methylation and three demonstrated significantly decreased methylation. In summary, the present study indicated that circRNA m6A methylation may be associated with pathogenesis of AD.

WTAP-mediated m6A modification modulates bone marrow mesenchymal stem cells differentiation potential and osteoporosis

An imbalance in the differentiation potential of bone marrow mesenchymal stem cells (BMSCs) is an important pathogenic mechanism underlying osteoporosis (OP). N6-methyladenosine (m6A) is the most common post-transcriptional modification in eukaryotic cells. The role of the Wilms’ tumor 1-associated protein (WTAP), a member of the m6A functional protein family, in regulating BMSCs differentiation remains unknown. We used patient-derived and mouse model-derived samples, qRT-PCR, western blot assays, ALP activity assay, ALP, and Alizarin Red staining to determine the changes in mRNA and protein levels of genes and proteins associated with BMSCs differentiation. Histological analysis and micro-CT were used to evaluate developmental changes in the bone. The results determined that WTAP promoted osteogenic differentiation and inhibited adipogenic differentiation of BMSCs. We used co-immunoprecipitation (co-IP), RNA immunoprecipitation (RIP), methylated RNA immunoprecipitation (MeRIP), RNA pulldown, and dual-luciferase assay to explore the direct mechanism. Mechanistically, the expression of WTAP increased during osteogenic differentiation and significantly promoted pri-miR-181a and pri-miR-181c methylation, which was recognized by YTHDC1, and increased the maturation to miR-181a and miR-181c. MiR-181a and miR-181c inhibited the mRNA expression of SFRP1, promoting the osteogenic differentiation of BMSCs. Our results demonstrated that the WTAP/YTHDC1/miR-181a and miR-181c/SFRP1 axis regulated the differentiation fate of BMSCs, suggesting that it might be a potential therapeutic target for osteoporosis.

Transcriptome-wide m6A modification mediates cardiotoxicity in mice after chronic exposure to microplastics

The widespread presence of microplastics (MPs) has garnered attention owing to their possible adverse effects. However, the cardiotoxicity of MPs and their underlying mechanism remains unclear. N6-methyladenosine (m6A) modification contributes to post-transcriptional modulation of gene expression, but whether this modification is relevant to MP-induced cardiotoxicity is unknown. First, we detected damage to the myocardial tissue among MP-exposed and -unexposed (control) mice by staining them with hematoxylin and eosin, Masson trichrome, and Oil Red O. Then, we comprehensively measured the transcriptome-wide m6A methylome and m6A-altered genes using high-throughput sequencing assays, such as methylated RNA immunoprecipitation sequencing and RNA sequencing. Our data indicated MP-induced myocardial inflammatory injury and histological alterations in vivo, evidenced by the severity of cardiac fibrosis and increased lipid accumulation. We found 878 increased and 316 decreased methylation peaks mostly distributed in the 3′ UTR among the MP-exposed group compared with the control group. We found 779 upregulated and 340 downregulated genes in the MP-exposed group. In addition, conjoint analysis of results from the two high-throughput sequencings showed that 109 and 11 hypermethylated genes were upregulated and downregulated, respectively; 12 and 21 hypomethylated genes were upregulated and downregulated, respectively. Results of the cross-link analysis showed that several potential signals, such as ECM–receptor interactions, cell adhesion molecules, cytokine–cytokine receptor interactions, and NF–κB signaling, contributed to MP-induced cardiotoxicity. Our findings indicated that m6A modifications of genes were involved in MP-induced cardiotoxicity and reported new information regarding the chronic cardiotoxicity caused by MP exposure in mice.

Identification and comparison of m6A modifications in glioblastoma non-coding RNAs with MeRIP-seq and Nanopore dRNA-seq

ABSTRACT The most prominent RNA modification – N6-methyladenosine (m6A) – affects gene regulation and cancer progression. The extent and effect of m6A on long non-coding RNAs (lncRNAs) is, however, still not clear. The most established method for m6A detection is methylated RNA immunoprecipitation and sequencing (MeRIP-seq). However, Oxford Nanopore Technologies recently developed direct RNA-seq (dRNA-seq) method, allowing m6A identification at higher resolution and in its native form. We performed whole transcriptome sequencing of the glioblastoma cell line U87-MG with both MeRIP-seq and dRNA-seq. For MeRIP-seq, m6A peaks were identified using nf-core/chipseq, and for dRNA-seq – EpiNano pipeline. MeRIP-seq analysis revealed 5086 lncRNAs transcripts, while dRNA-seq identified 336 lncRNAs transcripts from which 556 and 198 were found to be m6A modified, respectively. While 24 lncRNAs with m6A overlapped between two methods. Gliovis database analysis revealed that the expression of the major part of identified overlapping lncRNAs was associated with glioma grade or patient survival prognosis. We found that the frequency of m6A occurrence in lncRNAs varied more than 9-fold throughout the provided list of 24 modified lncRNAs. The highest m6A frequency was detected in MIR1915HG, THAP9-AS1, MALAT1, NORAD1, and NEAT1 (49–88nt), while MIR99AHG, SNHG3, LOXL1-AS1, ILF3-DT showed the lowest m6A frequency (445–261nt). Taken together, (1) we provide a high accuracy list of 24 m6A modified lncRNAs of U87-MG cells; (2) we conclude that MeRIP-seq is more suitable for an initial m6A screening study, due to its higher lncRNA coverage, whereas dRNA-seq is most useful when more in-depth analysis of m6A quantity and precise location is of interest. Abbreviations: (dRNA-seq) direct RNA-seq, (GBM) glioblastoma, (LGG) low-grade glioma, (lncRNAs) long non-coding RNAs, (m6A) N6-methyladenosine, (MeRIP-seq) methylated RNA immunoprecipitation and sequencing, (ncRNA) non-coding RNA, (ONT) Oxford Nanopore Technologi; Lietuvos Mokslo Taryba

Role of METTL3 in Aerobic Glycolysis of Glioma by Regulating m6A/miR-27b-3p/PDK1.

Methyltransferase like 3 (METTL3) has been reported to be dysregulated in glioma. However, its role in aerobic glycolysis of glioma remains unknown. This study was conducted to explore the molecular mechanism by which METTL3 regulates aerobic glycolysis of glioma and provide novel targets for the treatment of glioma. The expression levels of METTL3, microRNA (miR)-27b-3p, and pyruvate dehydrogenase kinase 1 (PDK1) were determined in glioma cell lines and normal human astrocytes. Cell proliferation and aerobic glycolysis were evaluated by cell counting kit-8 and colony formation assays and measurements of glucose uptake, lactate production, adenosine triphosphate, Hexokinase activity, oxygen consumption rate, and extracellular acidification rate. After m6A quantification analysis, methylated RNA immunoprecipitation, and the dual-luciferase assay, the rescue experiments were performed using miR-27b-3p inhibitor or pcDNA3.1-PDK1 with pcDNA3.1-METTL3. METTL3 was lower in glioma cells and METTL3 overexpression reduced aerobic glycolysis. METTL3 increased m6A modification to promote the processing of pri-miR-27b by DGCR8 and the expression of mature miR-27b-3p, and miR-27b-3p targeted and inhibited PDK1 expression. miR-27b-3p inhibition or PDK1 overexpression both neutralized the inhibitory role of METTL3 overexpression in aerobic glycolysis. Overall, METTL3 overexpression increased the expression of mature miR-27b-3p via m6A modification and inhibited PDK1 expression, thus suppressing aerobic glycolysis of glioma.

ALKBH5 alleviates hypoxia postconditioning injury in D-galactose-induced senescent cardiomyocytes by regulating STAT3.

Ischemic postconditioning (I/Post) reduces ischemia/reperfusion (I/R) injury by activating endogenous cardioprotection mechanisms, such as the JAK/STAT3 and PI3K/Akt pathways, which offer a traditional approach to myocardial protection. According to the previous study, cardioprotection by I/Post may lost in aged mice, and in our previous research, hypoxic postconditioning (H/Post) lacked a protective effect in senescent cardiomyocytes, which was associated with low expression of long noncoding RNA (lncRNA) H19. The N6-methyladenosine (m6A) modification is a dynamic and reversible process that has been confirmed plays a role in cardiovascular diseases. However, the mechanisms of m6A modification in myocardial I/Post remains to be explored. Neonatal cardiomyocytes were isolated from 2-day-old Sprague Dawley rats and senescence was induced by D-galactose, followed by stimulation of hypoxia-reoxygenation (H/R) and H/Post. Hypoxic injury was evaluated by cell viability and the Bcl-2/Bax protein ratio. Total m6A levels were measured using a colorimetric m6A RNA Methylation Quantification Kit and the m6A modified and differentially expressed mRNA was determined by methylated RNA immunoprecipitation (MeRIP). We found that H/Post increased m6A methylation and decreased RNA mA demethylase alkB homolog 5 (ALKBH5) expression in aged cardiomyocytes. Furthermore, ALKBH5 knockdown exacerbated injury following H/Post in senescent cardiomyocytes. Additionally, ALKBH5 regulated STAT3 expression by mediating its m6A modification and lncRNA H19/miR-124-3p. ALKBH5 also alleviated the H/Post injury induced by the low expression of STAT3 in senescent cardiomyocytes.

METTL3 Promotes the Growth and Invasion of Melanoma Cells by Regulating the lncRNA SNHG3/miR-330-5p Axis

Accumulating evidence indicates that m6A methyltransferase 3 (METTL3) plays a pivotal role in different malignancies including melanoma. However, the function and underlying mechanisms by which METTL3 contributes to the tumorigenesis of melanoma remain undocumented. The association of METTL3 and long noncoding RNA (lncRNA) small nucleolar RNA host gene 3 (SNHG3) with clinicopathological characteristics and prognosis in patients with melanoma was analyzed by real-time quantitative polymerase chain reaction (RT-qPCR), Western blotting, and The Cancer Genome Atlas data sets. The role of METTL3 in melanoma cells was assessed by in vitro and in vivo experiments. The m6A dot blot, methylated RNA immunoprecipitation (MeRIP), and RT-qPCR were used to verify METTL3-mediated m6A modification of lncRNA SNHG3. The effect of METTL3 on lncRNA SNHG3 was determined by luciferase gene reporter assay, RT-qPCR, and Western blotting. We found that METTL3 was upregulated in melanoma tissue samples and associated with poor survival in patients with melanoma. Knockdown of METTL3 suppressed the growth and invasion of melanoma cells in vitro and in vivo, whereas restored expression of METTL3 promoted these effects. Mechanistic investigations showed that knockdown of METTL3 reduced SNHG3 m6A levels and its messenger ribonucleic acid (mRNA) expression levels. SNHG3 could act as a sponge of microRNA (miR)-330-5p to upregulate the expression of CCHC-type zinc finger nucleic acid binding protein (CNBP). SNHG3 overexpression reversed METTL3-knockdown-caused antitumor effects, miR-330-5p upregulation and CNBP downregulation. SNHG3 had a positive correlation with METTL3 expression but a negative correlation with miR-330-5p expression in melanoma tissue samples. In conclusion, our findings demonstrated that METTL3-mediated m6A modification of lncRNA SNHG3 promoted the growth and invasion of melanoma cells by regulating the miR-330-5p/CNBP axis.

The m6A demethylase FTO promotes keloid formation by up-regulating COL1A1.

Keloid is a dermal fibrotic disease characterized by excessive proliferation of dermal fibroblasts and deposition of excessive collagen. N6-methyladenosine (m6A) plays a significant role in numerous physiological and pathological regulatory processes in the human body. Fat mass and obesity-associated protein (FTO) is one of the most essential m6A demethylases. However, whether FTO has a regulatory role in keloid development remains to be determined. In this study, we investigated the effects of the m6A demethylase FTO on keloid formation by performing hematoxylin and eosin (H&E) staining, m6A dot blotting, transwell migration experiment, and methylated RNA immunoprecipitation quantitative polymerase chain reaction (MeRIP-qPCR) tests, as well as real-time PCR (RT-PCR) and Western blot assays. The H&E staining indicated abnormal arrangement and proliferation of fibroblasts in the keloid tissue. The m6A dot blotting and qPCR revealed lower levels of m6A modification and increased expression of the m6A demethylases FTO in keloid tissue. Furthermore, overexpression of FTO promoted fibroblast migration as well as the expression of collagen type I alpha 1 chain (COL1A1) and α-smooth muscle actin (α-SMA). Mechanistic experiments demonstrated that FTO enhances keloid formation by modulating COL1A1 m6A modification and messenger RNA (mRNA) stability. In addition, this study also revealed the role of FTO in the therapeutic effect of glucocorticoids on keloids. Our study demonstrates that FTO upregulates COL1A1 expression via regulating COL1A1 m6A modification and maintaining mRNA stability, hence promoting keloid development and providing a potential new therapeutic target for the treatment of keloids.

Deubiquitinase USP13 regulates glycolytic reprogramming and progression in osteosarcoma by stabilizing METTL3/m6A/ATG5 axis.

Reprogramming metabolism is a hallmark of cancer cells for rapid progression. However, the detailed functional role of deubiquitinating enzymes (DUBs) in tumor glycolytic reprogramming is still unknown and requires further investigation. USP13 was found to upregulate in osteosarcoma (OS) specimens and promote OS progression through regulating aerobic glycolysis. Interestingly, the m6A writer protein, METTL3, has been identified as a novel target of USP13. USP13 interacts with, deubiquitinates, and therefore stabilizes METTL3 at K488 by removing K48-linked ubiquitin chains. Since METTL3 is a well-known m6A writer and USP13 stabilizes METTL3, we further found that USP13 increased global m6A abundance in OS cells. The results of RNA sequencing and methylated RNA immunoprecipitation sequencing indicated METTL3 could bind to m6A-modified ATG5 mRNA, which is crucial for autophagosome formation, and inhibit ATG5 mRNA decay on an IGF2BP3 dependent manner, thereby promoting autophagy and the autophagy-associated malignancy of OS. Using a small-molecule inhibitor named Spautin-1 to pharmacologically inhibit USP13 induced METTL3 degradation and exhibited significant therapeutic efficacy both in vitro and in vivo. Collectively, our study results indicate that USP13 promotes glycolysis and tumor progression in OS by stabilizing METTL3, thereby stabilizing ATG5 mRNA and facilitating autophagy in OS. Our findings demonstrate the role of the USP13-METTL3-ATG5 cascade in OS progression and show that USP13 is a crucial DUB for the stabilization of METTL3 and a promising therapeutic target for treating OS.

Variant rs8400 enhances ALKBH5 expression through disrupting miR-186 binding and promotes neuroblastoma progression.

AlkB homolog 5 (ALKBH5) has been proven to be closely related to tumors. However, the role and molecular mechanism of ALKBH5 in neuroblastomas have rarely been reported. The potential functional single-nucleotide polymorphisms (SNPs) in ALKBH5 were identified by National Center for Biotechnology Information (NCBI) dbSNP screening and SNPinfo software. TaqMan probes were used for genotyping. A multiple logistic regression model was used to evaluate the effects of different SNP loci on the risk of neuroblastoma. The expression of ALKBH5 in neuroblastoma was evaluated by Western blotting and immunohistochemistry (IHC). Cell counting kit-8 (CCK-8), plate colony formation and 5-ethynyl-2'-deoxyuridine (EdU) incorporation assays were used to evaluate cell proliferation. Wound healing and Transwell assays were used to compare cell migration and invasion. Thermodynamic modelling was performed to predict the ability of miRNAs to bind to ALKBH5 with the rs8400 G/A polymorphism. RNA sequencing, N6-methyladenosine (m6A) sequencing, m6A methylated RNA immunoprecipitation (MeRIP) and a luciferase assay were used to identify the targeting effect of ALKBH5 on SPP1. ALKBH5 was highly expressed in neuroblastoma. Knocking down ALKBH5 inhibited the proliferation, migration and invasion of cancer cells. miR-186-3p negatively regulates the expression of ALKBH5, and this ability is affected by the rs8400 polymorphism. When the G nucleotide was mutated to A, the ability of miR-186-3p to bind to the 3'-UTR of ALKBH5 decreased, resulting in upregulation of ALKBH5. SPP1 is the downstream target gene of the ALKBH5 oncogene. Knocking down SPP1 partially restored the inhibitory effect of ALKBH5 downregulation on neuroblastoma. Downregulation of ALKBH5 can improve the therapeutic efficacy of carboplatin and etoposide in neuroblastoma. We first found that the rs8400 G>A polymorphism in the m6A demethylase-encoding gene ALKBH5 increases neuroblastoma susceptibility and determines the related mechanisms. The aberrant regulation of ALKBH5 by miR-186-3p caused by this genetic variation in ALKBH5 promotes the occurrence and development of neuroblastoma through the ALKBH5-SPP1 axis.

CUCUME: An RNA methylation database integrating systemic mRNAs signals, GWAS and QTL genetic regulation and epigenetics in different tissues of Cucurbitaceae

As an internal modification of transcripts, RNA methylation determines RNA fate by changing RNA–protein binding affinity. In plants, RNA methylation is ubiquitous and is involved in all aspects of RNA post-transcriptional regulation. For instance, long-distance mobile RNAs, strongly influenced by their methylation status, play important roles in plant growth, development and environmental adaptation. Cucumber/pumpkin heterografts are widely used to improve stress tolerance of cucumber and to study mobile RNA signals due to their strong developed vasculature system. Here, we developed the Cucume (Cucurbit RNA methylation, http://cucume.cn/) database for these two important vegetables, cucumber (Cucumis sativus L.) and pumpkin (Cucurbita moschata) with high productivity worldwide. We identified mRNAs harboring 5-methylcytosine (m5C) and N6-methyladenosine (m6A) sites in pumpkin and cucumber at the whole genome level via Methylated RNA Immunoprecipitation sequencing (MeRIP-seq) of different tissues and the vascular exudates. In addition to RNA methylation sites, the Cucume database includes graft-transmissible systemic mRNAs identified in previous studies using cucumber/pumpkin heterografts. The further integration of cucumber genome-wide association analysis (GWAS) and quantitative trait loci (QTL) allows the study of RNA methylation-related genetic and epigenetic regulation in cucurbits. Therefore, the here developed Cucume database will promote understanding the role of cucurbit RNA methylation in RNA mobility and QTL, ultimately benefitting future breeding of agronomic crop germplasms.

METTL14 Facilitates the Metastasis of Pancreatic Carcinoma by Stabilizing LINC00941 in an m6A-IGF2BP2-Dependent Manner.

Pancreatic adenocarcinoma (PC), one of the most fatal diseases, usually generates a poor prognosis in advanced stages. N6-methyladenosine modification has emerged as a crucial participant in tumor development and recurrence. Methyltransferase-like 14 (METTL14), as a core member of methyltransferases, is involved in tumor progression and metastasis. However, the potential mechanism by which METTL14 regulates long noncoding RNAs (lncRNAs) in PC remains unclear. RNA immunoprecipitation (RIP), methylated RNA immunoprecipitation quantitative PCR (MeRIP-qPCR), and fluorescence in situ hybridization (FISH) were used to explore the underlying mechanisms. In our study, we found that METTL14 expression was upregulated in PC patients, and was associated with poor prognosis. In vitro and in vivo experiments, knocking down METTL14 suppressed tumor metastasis. RNA-seq and bioinformatics analyses were used to identify LINC00941 as the downstream target of METTL14. Mechanistically, LINC00941 was upregulated by METTL14 in an m6A-dependent way. LINC00941 was recruited and recognized by IGF2BP2. METTL14 enhanced the affinity of IGF2BP2 for LINC00941, while IGF2BP2 promoted the stabilization of LINC00941, which contributed to the migration and invasion of PC cells. Overall, our research revealed that METTL14 promoted the metastasis of PC through m6A modification of LINC00941. Targeting the METTL14-LINC00941-IGF2BP2 axis may provide promising therapeutic approaches for PC.

AZGP1P2/UBA1/RBM15 Cascade Mediates the Fate Determinations of Prostate Cancer Stem Cells and Promotes Therapeutic Effect of Docetaxel in Castration-Resistant Prostate Cancer via TPM1 m6A Modification.

Prostate cancer (PCa) is a common malignant tumor with high morbidity and mortality worldwide. The prostate cancer stem cell (PCSC) model provides novel insights into the pathogenesis of PCa and its therapeutic response. However, the roles and molecular mechanisms of specific genes in mediating fate decisions of PCSCs and carcinogenesis of PCa remain to be elusive. In this study, we have explored the expression, function, and mechanism of AZGP1P2, a pseudogene of AZGP1, in regulating the stemness and apoptosis of PCSCs and treatment resistance of docetaxel in castration-resistant prostate cancer (CRPC). We revealed that AZGP1P2 was downregulated in CRPC cell lines and PCSCs, while it was positively associated with progression-free interval. Upregulation of the AZGP1P2 enhanced the sensitivity of docetaxel treatment in CRPCs via inhibiting their stemness. RNA pull-down associated with mass spectrometry analysis, co-immunoprecipitation assay, and RNA immunoprecipitation assay demonstrated that AZGP1P2 could bind to UBA1 and RBM15 as a "writer" of methyltransferase to form a compound. UBA1, an E1 ubiquitin-activating enzyme, contributed to RBM15 protein degradation via ubiquitination modification. Methylated RNA immunoprecipitation assay displayed that RBM15 controlled the mRNA decay of TPM1 in m6A methylation. Furthermore, a xenograft mouse model and patient-derived organoids showed that the therapeutic effect of docetaxel in CRPC was increased by AZGP1P2 invivo. Collectively, these results imply that AZGP1P2 mediates the stemness and apoptosis of PCSCs and promotes docetaxel therapeutic effect by suppressing tumor growth and metastasis via UBA1/RBM15-mediated TPM1 mRNA decay in CRPC.

Integrative analysis implicates the significance of m6A in the liver fibrosis of biliary atresia by regulating THY1.

Whether N6-methyladenosine (m6A) is involved in biliary atresia (BA) remains undefined. Herein, we comprehensively evaluated the m6A profile in BA. When compared with normal controls, BA had an elevated m6A level with upregulated m6A writers. The m6A level was correlated with liver function, stage of fibrosis and jaundice clearance in BA. Methylated RNA immunoprecipitation sequencing (MeRIP-seq) demonstrated an altered m6A topology in BA. MeRIP-seq and RNA sequencing filtered out 130 m6A-modified genes, which were enriched in fibrogenetic pathways. MeRIP-qPCR in vivo and interventions of LX-2 and primary HSCs in vitro validated the regulatory role of m6A on COL1A1 and THY1. THY1+ myofibroblasts expanded in portal area of BA, and highly expressed profibrogenic genes (COL1A1, MMP2, PDGFRA, and DCN). THY1 was correlated with liver fibrosis and jaundice clearance in BA. Bulk array (GSE46960, GSE15235), single-cell RNA sequencing (GSE136103), primary HSC interventions, and co-immunoprecipitation revealed that THY1 was correlated with extracellular matrix organization, promoted HSC activation, showed higher interactions with integrins on myeloid cells in cholestatic fibrosis, and was correlated with native liver survival in BA. Our study highlights the significance of m6A in BA-induced liver fibrogenesis by regulating THY1, shedding new light on the novel therapies to alleviate liver fibrosis by targeting m6A/THY1 axis in BA.

MFAP2, upregulated by m1A methylation, promotes colorectal cancer invasiveness via CLK3.

Distant metastasis is the main cause of mortality in colorectal cancer (CRC) patients. N1-methyladenosine (m1A) is a type of epitranscriptome modification. While its regulatory effect on mRNA and its role in CRC metastasis remain unclear. The m1A methylation profile of mRNAs in CRC was revealed by m1A methylated RNA immunoprecipitation sequencing. The expression of MFAP2 in tumor tissues was measured by immunohistochemistry and then correlated with the clinical characteristics and prognosis of CRC patients. The role of MFAP2 in the invasiveness of CRC cells was evaluated by transwell assays and peritoneal metastatic model in nude mice. The downstream targets of MFAP2 was screened by mass spectrometry analysis. Then the role of MFAP2-CLK3 signaling axis was verified by cotransfecting MFAP2 siRNA and CLK3 plasmid in CRC cells. Microfibril associated protein 2 (MFAP2) mRNA was overexpressed and m1A-hypermethylated in CRC. High expression of MFAP2 was closely related to lymph node metastasis and distant metastasis, leading to poor prognosis in patients with CRC. In vivo and in vitro studies showed that silencing of MFAP2 inhibited the migration, invasion and metastasis of CRC cells. CDC Like Kinase 3 (CLK3) was a potential downstream target of MFAP2. Further studies showed that MFAP2 depletion might induce autophagic degradation of CLK3, and the role of MFAP2 in the invasiveness of CRC cells was dependent on CLK3. Our results uncover a newly identified MFAP2-CLK3 signaling axis, which is a potential therapeutic target for CRC metastasis.

Integrated Profiles of Transcriptome and mRNA m6A Modification Reveal the Intestinal Cytotoxicity of Aflatoxin B1 on HCT116 Cells.

Aflatoxin B1 (AFB1) is widely prevalent in foods and animal feeds and is one of the most toxic and carcinogenic aflatoxin subtypes. Existing studies have proved that the intestine is targeted by AFB1, and adverse organic effects have been observed. This study aimed to investigate the relationship between AFB1-induced intestinal toxicity and N6-methyladenosine (m6A) RNA methylation, which involves the post-transcriptional regulation of mRNA expression. The transcriptome-wide m6A methylome and transcriptome profiles in human intestinal cells treated with AFB1 are presented. Methylated RNA immunoprecipitation sequencing and mRNA sequencing were carried out to determine the distinctions in m6A methylation and different genes expressed in AFB1-induced intestinal toxicity. The results showed that there were 2289 overlapping genes of the differentially expressed mRNAs and differentially m6A-methylation-modified mRNAs. After enrichment of the signaling pathways and biological processes, these genes participated in the terms of the cell cycle, endoplasmic reticulum, tight junction, and mitophagy. In conclusion, the study demonstrated that AFB1-induced HCT116 injury was related to the disruptions to the levels of m6A methylation modifications of target genes and the abnormal expression of m6A regulators.

Chronic allergic asthma alters m6A epitranscriptomic tagging of mRNAs and lncRNAs in the lung.

To evaluate the role of m6A methylation of mRNAs and long non-coding RNAs (lncRNAs) in chronic allergic asthma. Transcriptome-wide N6-methyladenosine (m6A) changes in BALB/c mice were profiled using immunoprecipitated methylated RNAs with microarrays in lung with chronic allergic asthma. Gene ontology (GO) and KEGG analyses were conducted. Target genes were verified by methylated RNA immunoprecipitation and real-time polymerase chain reaction (PCR). Specifically, the mRNA levels of m6A writers (METTL3, METTL14, and WTAP), and readers and erasers (FTO and ALKBH5) were estimated by real-time PCR analysis, using the SYBR-green method. IL17RB mRNA was also evaluated by PCR. Hematoxylin and eosin (H&E) staining showed that the airway and lung tissues in mice in the asthma group had extensive infiltration of inflammatory cells around the bronchioles, blood vessels, and alveoli. The lungs of those allergic asthma mice showed altered m6A epitranscriptome, whereby 1369 mRNAs and 176 lncRNAs were hypermethylated, and 197 mRNAs and 30 lncRNAs were hypomethylated (>1.5-fold vs control). Also, compared with the control group, IL17RB mRNA in lung of the asthmatic group was significantly hypermethylated (P<0.01). In the asthma group, the mRNA and the protein level of METTL14 (the key methyltransferase) and ALKBH5 (the major demethyltransferase) were significantly decreased compared with the control group (P<0.01). Chronic allergic asthma alters the lung m6A epitranscriptome, suggesting functional implications in the pathophysiology of refractory asthma. Data support methylated IL17RB mRNA possibly becoming a new therapeutic target for chronic allergic asthma.