m6A Levels Research Articles

Molecular mechanism of ALKBH5-mediated m6A demethylation regulating lipopolysaccharide-induced epithelial-mesenchymal transition in sepsis-induced acute kidney injury.

This study explored the mechanism by which the m6A demethylase ALKBH5 mediates epithelial-mesenchymal transition (EMT) in sepsis-associated acute kidney injury (SA-AKI) and AKI-chronic kidney disease (CKD) transition. HK-2 cells were stimulated with lipopolysaccharide (LPS) to establish an in vitro model of SA-AKI. ALKBH5 expression was reduced through the transfection of si-ALKBH5. Cell viability, apoptosis, and migration were detected by CCK-8 assay, TUNEL staining, and Transwell. The levels of TNF-α, IL-1β, and IL-6 were measured by enzyme-linked immunosorbent assay. Quantitative real-time polymerase chain reaction or Western blotting was performed to determine the expressionsof ALKBH5, miR-205-5p, DDX5, E-cadherin, and α-SMA. The m6A level was quantitatively analyzed. The expression of pri-miR-205 bound to DGCR8 and m6A-modified pri-miR-205 after intervention with ALKBH5 expression was detected by RNA immunoprecipitation. A dual-luciferase assay confirmed the binding between miR-205-5p and DDX5. ALKBH5 was highly expressed in LPS-induced HK-2 cells. Inhibition of ALKBH5 increased cell viability, repressed apoptosis, and reduced EMT. Inhibition of ALKBH5 increased the m6A modification level, thereby promoting DGCR8 binding to pri-miR-205 to increase miR-205-5p expression and eventually targeting DDX5 expression. Low expression of miR-205-5p or overexpression of DDX5 partially abolished the inhibitory effect of ALKBH5 silencing on EMT. In conclusion, ALKBH5 represses miR-205-5p expression by removing m6A modification to upregulate DDX5 expression, thereby promoting EMT and AKI-CKD transition after SA-AKI.

Ligation-driven hyperbranched DNA assembly for label-free and sequence-specific measurement of RNA N6-methyladenosine in human breast tissues

N6-methyladenosine (m6A) is a critical post-transcriptional regulator, and it is linked to various biological functions (e.g., degradation, export, and translation) and multiple human diseases (e.g., cancers, immune disorders, and neurodegenerative diseases). The current methods for sequence-specific detection of m6A usually suffer from radioactive risk, large sample consumption, washing/separation steps, and false-positive signal. Herein, we develop a ligation-driven hyperbranched DNA assembly-based fluorescent biosensor for label-free and sequence-specific detection of m6A. In this assay, the unmethylated RNA can be identified and cleaved by m6A-sensitive endoribonuclease MazF. The retained intact m6A-RNA is complementary to the padlock probe to form a circular DNA, subsequently initiating ligation-driven hyperbranched rolling circle amplification (HRCA). The obtained HRCA products can be simply quantified with SYBR Gold as a fluorescent indicator. This method can be homogeneously carried out in an isothermal, label-free and antibody-free manner. The limit of detection can reach 0.079 pM. Moreover, this method can differentiate even 0.01 % m6A level in excess coexisting interferences, monitor cellular m6A RNA level with single-cell sensitivity, and differentiate m6A level in breast tumor tissues and normal adjacent tissues, with promising applications in m6A-releated epigenetic research and clinical diagnosis.

METTL3-driven m6A modification of lncRNA FAM230B suppresses ferroptosis by modulating miR-27a-5p/BTF3 axis in gastric cancer

Our previous research revealed the apoptosis-inhibiting effect of lncRNA FAM230B in gastric cancer (GC). While its role on ferroptosis of GC remain unexplored. In this study, the m6A level and RNA stability regulation of METTL3 on FAM230B was detected by m6A quantification, stability assays, MeRIP, and their interaction was confirmed by RIP, and RNA pull-down assays. The level of ferroptosis was detected by flow cytometry, MDA and GSH level assessments, and electron microscopy. Gene expression was detected by quantitative real-time PCR, western blot, and immunofluorescence. The miR-27a-5p and BTF3 interaction was predicted with TargetScan and confirmed by dual-luciferase assay. Here, elevated levels of METTL3 and FAM230B were observed in GC tissues and cell lines. METTL3 was confirmed to bind with FAM230B RNA. Furthermore, silencing METTL3 reduced FAM230B m6A levels and stability, leading to decreased FAM230B and increased miR-27a-5p expressions. FAM230B knockdown favored ferroptosis and increased BTF3 expression, while its overexpression mitigated erastin-induced ferroptosis in GC cells. Additionally, BTF3 overexpression was found to negate miR-27a-5p's ferroptosis-promoting effects in GC cells. Collectively, our study demonstrates that the m6A modification of FAM230B by METTL3 plays a crucial role in promoting GC progression by reducing ferroptosis, through the modulation of the miR-27a-5p/BTF3 axis.

METTL3-Mediated m6A Modification of FMRP Drives Hepatocellular Carcinoma Progression and Indicates Poor Prognosis.

Accumulating studies reveal that m6A RNA methylation plays a critical role in cancer pathogenesis and progression. METTL3 as a m6A methyltransferase acts as an oncogene in multiple malignancies including hepatocellular carcinoma (HCC). However, the role and underlying mechanism by which METTL3 contributes to HCC remain unclear. The association of METTL3 expression with clinicopathological characteristics and prognosis in patients with HCC was assessed by reverse transcription polymerase chain reaction, Western blot, and public TCGA dataset. MTT, colony formation, Transwell assays, and xenograft tumor models were executed to reveal the role of METTL3 in HCC. m6A dot blot, RNA immunoprecipitation (RIP), m6A methylated RIP, and Western blot assays were used to uncover the regulatory mechanism of METTL3 in HCC cells. We found that METTL3 was dramatically upregulated in HCC tissue samples and acted as an independent prognostic factor for poor survival and tumor recurrence in patients with HCC. Silencing of METTL3 repressed cell growth and invasion in vitro and invivo, but restored expression of METTL3 boosted these effects. Mechanistical investigations revealed that METTL3 could directly interact with FMRP and harbor a positive correlation with FMRP expression. Knockdown of METTL3 reduced FMRP m6A levels as well as its mRNA and protein expression. FMRP overexpression drove cell colony formation and cell invasion and abolished METTL3 knockdown-induced antitumor effects and AKT/mTORC1 signaling inactivation. Elevated expression of FMRP could act as an independent prognostic factor for poor survival and tumor recurrence in patients with HCC. Our findings demonstrate that METTL3-mediated m6A modification of FMRP promotes growth and invasion of HCC cells and may provide a promising therapeutic target for HCC.

METTL3 silencing inhibits ferroptosis to suppress ovarian fibrosis in PCOS by upregulating m6A modification of GPX4.

Methyltransferase-like 3 (METTL3) is extensively reported to be involved in organ fibrosis. Ovarian fibrosis is a main characteristic of polycystic ovary syndrome (PCOS). However, the reaction mechanism of METTL3 in PCOS is poorly investigated. This paper was intended to reveal the role and the mechanism of METTL3 in PCOS. Animal and cell models of PCOS were induced by dehydroepiandrosterone (DHEA). H&E staining was performed to detect the pathological alterations in ovary tissues. Masson staining, immunofluorescence, along with western blot measured fibrosis both in vitro and in vivo. To evaluate estrous cycle, vaginal smear was performed. Lipid peroxidation and ferroptosis were evaluated by MDA assay kits, GSH assay kits, immunohistochemistry, Prussian blue staining and western blot. qRT-PCRand western blot were adopted to estimate METTL3 and GPX4 expression. The m6A and hormone secretion levels were respectively assessed by m6A RNA Methylation Quantitative Kit and corresponding kits. The interaction between METTL3 and GPX4 was testified by immunoprecipitation. The fibrosis and ferroptosis were aggravated and m6A and METTL3 expression were increased in ovarian tissues of DHEA-induced PCOS mice. METTL3 silencing alleviated pathological changes, affected hormone secretion level, and repressed fibrosis, lipid peroxidation and ferroptosis in the ovarian tissues of PCOS mice. In vitro, DHEA stimulation increased m6A and METTL3 expression and induced ferroptosis and fibrosis. METTL3 knockdown promoted GPX4 expression in DHEA-induced granulosa cells by m6A modification and restrained DHEA-induced fibrosis, lipid peroxidation and ferroptosis in granulosa cells via elevating GPX4. METTL3 silence inhibited ovarian fibrosis in PCOS, which was mediated through suppressing ferroptosis by upregulating GPX4 in m6A-dependent manner.

HucMSC-Ex alleviates inflammatory bowel disease in mice by enhancing M2-type macrophage polarization via the METTL3-Slc37a2-YTHDF1 axis

Human umbilical cord mesenchymal stem cell-derived exosomes (hucMSC-Ex) have emerged as a new treatment strategy for inflammatory bowel disease (IBD) due to their immunoregulatory function. N6-methyladenosine (m6A) plays a crucial role in regulating intestinal immunity, especially in IBD where macrophages play an important role, although its mechanism is not yet fully understood. From this perspective, this research aimed to evaluate the effect of hucMSC-Ex on m6A modification of macrophages in IBD. In the process of alleviating inflammation, hucMSC-Ex promotes macrophage polarization toward the M2 type and regulates intracellular m6A levels by upregulating the expression of m6A “Writer” METTL3 and “Reader” YTHDF1. Solute Carrier Family 37 Member 2 (Slc37a2) was identified by Methylation RNA immunoprecipitation sequencing as the target molecule of the hucMSC-Ex. Mechanically, hucMSC-Ex promoted the binding of METTL3 to the Slc37a2 mRNA complex, and enhanced the binding of Slc37a2 to YTHDF1 to upregulate the intracellular expression of Slc37a2, thereby attenuating the pro-inflammatory function of macrophage. This study confirms the modulatory role of hucMSC-Ex on the m6A modification of macrophages in IBD, providing a new scientific basis for the treatment of IBD with hucMSC-Ex.

The m6A writer KIAA1429 regulates photoaging progression via MFAP4-dependent collagen synthesis

BackgroundN6-Methyladenosine (m6A) methylation, a common form of RNA modification, play an important role in the pathogenesis of various diseases and in the ontogeny of organisms. Nevertheless, the precise function of m6A methylation in photoaging remains unknown.ObjectivesThis study aims to investigate the biological role and underlying mechanism of m6A methylation in photoaging.Methodsm6A dot blot, Real-time quantitative PCR (RT-qPCR), western blot and immunohistochemical (IHC) assays were employed to detect the m6A level and specific m6A methylase in ultraviolet ray (UVR)-induced photoaging tissue. The profile of m6A-tagged mRNA was identified by methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA-seq analysis. Finally, we investigated the regulatory mechanism of KIAA1429 by MeRIP-qPCR, RNA knockdown and immunofluorescence assay.Resultsm6A levels were increased in photoaging and were closely associated with the upregulation of KIAA1429 expression. 1331 differentially m6A methylated genes were identified in the UVR group compared with the control group, of which 1192 (90%) were hypermethylated. Gene ontology analysis showed that genes with m6A hypermethylation and mRNA downregulation were mainly involved in extracellular matrix metabolism and collagen metabolism-related processes. Furthermore, KIAA1429 knockdown abolished the downregulation of TGF-bRII and upregulation of MMP1 in UVR-irradiated human dermal fibroblasts (HDFs). Mechanically, we identified MFAP4 as a target of KIAA1429-mediated m6A modification and KIAA1429 might suppress collagen synthesis through an m6A-MFAP4-mediated process.ConclusionsThe increased expression of KIAA1429 hinders collagen synthesis during UVR-induced photoaging, suggesting that KIAA1429 represents a potential candidate for targeted therapy to mitigate UVR-driven photoaging.

Epitranscriptomic regulation of lipid oxidation and liver fibrosis via ENPP1 mRNA m6A modification

BackgroundDysregulated lipid oxidation occurs in several pathological processes characterized by cell proliferation and migration. Nonetheless, the molecular mechanism of lipid oxidation is not well appreciated in liver fibrosis, which is accompanied by enhanced fibroblast proliferation and migration.MethodsWe investigated the causes and consequences of lipid oxidation in liver fibrosis using cultured cells, animal models, and clinical samples.ResultsIncreased ecto-nucleotide pyrophosphatase/phosphodiesterase (ENPP1) expression caused increased lipid oxidation, resulting in the proliferation and migration of hepatic stellate cells (HSCs) that lead to liver fibrosis, whereas fibroblast-specific ENPP1 knockout reversing these results. Elevated ENPP1 and N6-methyladenosine (m6A) levels were associated with high expression of Wilms tumor 1 associated protein (WTAP). Mechanistically, WTAP-mediated m6A methylation of the 3'UTR of ENPP1 mRNA and induces its translation dependent of YTH domain family proteins 1 (YTHDF1). Additionally, ENPP1 could interact with hypoxia inducible lipid droplet associated (HILPDA) directly; overexpression of ENPP1 further recruits HILPDA-mediated lipid oxidation, thereby promotes HSCs proliferation and migration, while inhibition of ENPP1 expression produced the opposite effect. Clinically, increased expression of WTAP, YTHDF1, ENPP1, and HILPDA, and increased m6A mRNA content, enhanced lipid oxidation, and increased collagen deposition in human liver fibrosis tissues.ConclusionsWe describe a novel mechanism in which WTAP catalyzes m6A methylation of ENPP1 in a YTHDF1-dependent manner to enhance lipid oxidation, promoting HSCs proliferation and migration and liver fibrosis.Graphical abstract

Biogenesis of circRBM33 mediated by N6-methyladenosine and its function in abdominal aortic aneurysm

ABSTRACT This study aimed to explore whether m6A modification affects the biogenesis of circRBM33, which is involved in the progression of abdominal aortic aneurysm (AAA). For in vitro experiments, vascular smooth muscle cells (VSMCs) were treated with Ang II. MeRIP‒PCR was used to assess m6A modification of circRBM33. Gene expression was measured using RT‒qPCR and Western blotting. For in vivo experiments, a mouse model of AAA was established via Ang II infusion. HE, Sirius Red and TUNEL staining was performed to evaluate pathological changes and cell apoptosis in aortic vessels. The results showed that the m6A level of circRBM33 was abnormally increased in Ang II-induced VSMCs. In addition, METTL3 positively regulated circRBM33 expression. YTHDC1 deficiency decreased circRBM33 expression but had no effect on RBM33 mRNA expression. Notably, neither METTL3 nor YTHDC1 influenced the stability of circRBM33 or RBM33 mRNA. The interaction between circRBM33 and METTL3/YTHDC1 was verified by RIP analysis. Moreover, the Ang II-induced increase in circRBM33 expression was reversed by cycloleucine (an inhibitor of m6A methylation). Importantly, the m6A modification and expression of circRBM33 in the circRBM33-m6A-mut2-expressing VSMCs were not altered by METTL3 silencing. Mechanistically, METTL3/YTHDC1 modulates the biogenesis of circRBM33 in an m6A-dependent manner. In addition, circRBM33 knockdown alleviated AAA by reducing ECM degradation in the Ang II-infused mice. In conclusion, this study demonstrated that METTL3/YTHDC1-mediated m6A modification modulates the biogenesis of circRBM33 from exons of the RBM33 gene. Moreover, knockdown of circRBM33 alleviated AAA by reducing ECM degradation, which may provide a novel therapeutic strategy for treating AAA.

Genome-Wide Identification of the Soybean AlkB Homologue Gene Family and Functional Characterization of GmALKBH10Bs as RNA m6A Demethylases and Expression Patterns under Abiotic Stress.

Soybean (Glycine max (L.) Merr) is one of the most important crops worldwide, but its yield is vulnerable to abiotic stresses. In Arabidopsis, the AlkB homologue (ALKBH) family genes plays a crucial role in plant development and stress response. However, the identification and functions of its homologous genes in soybean remain obscured. Here, we identified a total of 22 ALKBH genes in soybean and classified them into seven subfamilies according to phylogenetic analysis. Gene duplication events among the family members and gene structure, conserved domains, and motifs of all candidate genes were analyzed. By comparing the changes in the m6A levels on mRNA from hair roots between soybean seedlings harboring the empty vector and those harboring the GmALKBH10B protein, we demonstrated that all four GmALKBH10B proteins are bona fide m6A RNA demethylases in vivo. Subcellular localization and expression patterns of the GmALKBH10B revealed that they might be functionally redundant. Furthermore, an analysis of cis-elements coupled with gene expression data demonstrated that GmALKBH10B subfamily genes, including GmALKBH10B1, GmALKBH10B2, GmALKBH10B3, and GmALKBH10B4, are likely involved in the cis-elements' response to various environmental stimuli. In summary, our study is the first to report the genome-wide identification of GmALKBH family genes in soybean and to determine the function of GmALKBH10B proteins as m6A RNA demethylases, providing insights into GmALKBH10B genes in response to abiotic stresses.

CRISPR/dCas13(Rx) Derived RNA N6-methyladenosine (m6A) Dynamic Modification in Plant.

N6-methyladenosine (m6A) is the most prevalent internal modification of mRNA and plays an important role in regulating plant growth. However, there is still a lack of effective tools to precisely modify m6A sites of individual transcripts in plants. Here, programmable m6A editing tools are developed by combining CRISPR/dCas13(Rx) with the methyltransferase GhMTA (Targeted RNA Methylation Editor, TME) or the demethyltransferase GhALKBH10 (Targeted RNA Demethylation Editor, TDE). These editors enable efficient deposition or removal of m6A modifications at targeted sites of endo-transcripts GhECA1 and GhDi19 within a broad editing window ranging from 0 to 46nt. TDE editor significantly decreases m6A levels by 24%-76%, while the TME editor increases m6A enrichment, ranging from 1.37- to 2.51-fold. Furthermore, installation and removal of m6A modifications play opposing roles in regulating GhECA1 and GhDi19 mRNA transcripts, which may be attributed to the fact that their m6A sites are located in different regions of the genes. Most importantly, targeting the GhDi19 transcript with TME editor plants results in a significant increase in root length and enhanced drought resistance. Collectively, these m6A editors can be applied to study the function of specific m6A modifications and have the potential for future applications in crop improvement.

METTL3 inhibitor suppresses the progression of prostate cancer via IGFBP3/AKT pathway and synergizes with PARP inhibitor

The RNA N6-methyladenosine (m6A) regulator METTL3 is an important regulatory gene in various progressive processes of prostate cancer (PCa). METTL3 inhibitors have been reported to possess potent tumor suppression capacity in some cancer types. Nevertheless, the detailed influence and mechanism of METTL3 inhibitors on PCa progression and their potential synergy with other drugs are poorly understood. In this study, we demonstrated that METTL3 was overexpressed and associated with poor survival in most PCa patients. METTL3 inhibitor STM2457 reduced m6A levels of PCa cells, thus inhibiting their proliferation, colony formation, migration, invasion, and stemness in vitro. Furthermore, STM2457 suppressed PCa progression in both the CDX and PDX models in vivo. MeRIP-seq analysis coupled with biological validation revealed that STM2457 influenced multiple biological processes in PCa cells, mainly through the IGFBP3/AKT pathway. We also proved that STM2457 induced DNA damage and showed synergistic anti-PCa effects with the PARP inhibitor olaparib both in vitro and in vivo. All in all, this work provides a novel therapeutic strategy for targeting RNA m6A modifications for the treatment of PCa and provides a meaningful reference for further clinical trials.

One-carbon metabolism supports S-adenosylmethionine and m6A methylation to control the osteogenesis of bone marrow stem cells and bone formation.

The skeleton is a metabolically active organ undergoing continuous remodeling initiated by bone marrow stem cells (BMSCs). Recent research has demonstrated that BMSCs adapt the metabolic pathways to drive the osteogenic differentiation and bone formation, but the mechanism involved remains largely elusive. Here, using a comprehensive targeted metabolome and transcriptome profiling, we revealed that one-carbon metabolism was promoted following osteogenic induction of BMSCs. Methotrexate (MTX), an inhibitor of one-carbon metabolism that blocks S-adenosylmethionine (SAM) generation, led to decreased N6-methyladenosine (m6A) methylation level and inhibited osteogenic capacity. Increasing intracellular SAM generation through betaine addition rescued the suppressed m6A content and osteogenesis in MTX-treated cells. Using S-adenosylhomocysteine (SAH) to inhibit the m6A level, the osteogenic activity of BMSCs was consequently impeded. We also demonstrated that the pro-osteogenic effect of m6A methylation mediated by one-carbon metabolism could be attributed to HIF-1α and glycolysis pathway. This was supported by the findings that dimethyloxalyl glycine rescued the osteogenic potential in MTX-treated and SAH-treated cells by upregulating HIF-1α and key glycolytic enzymes expression. Importantly, betaine supplementation attenuated MTX-induced m6A methylation decrease and bone loss via promoting the abundance of SAM in rat. Collectively, these results revealed that one-carbon metabolite SAM was a potential promoter in BMSC osteogenesis via the augmentation of m6A methylation, and the cross talk between metabolic reprogramming, epigenetic modification, and transcriptional regulation of BMSCs might provide strategies for bone regeneration.

Dynamic m6A mRNA methylation reveals the involvement of AcALKBH10 in ripening-related quality regulation in kiwifruit.

Kiwifruit ripening is a complex and highly coordinated process that occurs in conjunction with the formation of fruit edible quality. The significance of epigenetic changes, particularly the impact of N6-methyladenosine (m6A) RNA modification on fruit ripening and quality formation, has been largely overlooked. We monitored m6A levels and gene expression changes in kiwifruit at four different stages using LC-MS/MS, MeRIP, RNA-seq, and validated the function of AcALKBH10 through heterologous transgenic expression in tomato. Notable m6A modifications occurred predominantly at the stop codons and the 3' UTRs and exhibited a gradual reduction in m6A levels during the fruit ripening process. Moreover, these m6A modifications in the aforementioned sites demonstrated a discernible inverse relationship with the levels of mRNA abundance throughout the ripening process, suggesting a repression effect of m6A modification in the modulation of kiwifruit ripening. We further demonstrated that AcALKBH10 rather than AcECT9 predominantly regulates m6A levels in ripening-related genes, thereby exerting the regulatory control over the ripening process and the accumulation of soluble sugars and organic acids, ultimately influencing fruit ripening and quality formation. In conclusion, our findings illuminate the epi-regulatory mechanism involving m6A in kiwifruit ripening, offering a fresh perspective for cultivating high-quality kiwifruit with enhanced nutritional attributes.

Quantification of m6A RNA modification in Caenorhabditis elegans by LC‐MS

AbstractN6‐methyladenosine (m6A), one of the most abundant RNA modifications, has been found to be present in RNA in various life forms and plays a crucial regulatory role in gene expression. Caenorhabditis elegans (C. elegans) is a unique model organism with abundant m6A modifications. To analyze and understand the function of m6A in C. elegans, mass spectrometry is preferred because of its remarkable sensitivity and accuracy in detecting modified nucleosides. Here, we present a protocol for quantifying adenosine and m6A levels in C. elegans RNA. Using nucleases and phosphatases, we digested RNA into nucleosides and then quantified it using liquid chromatography‐mass spectrometry (LC–MS).

WTAP participates in neuronal damage by protein translation of NLRP3 in an m6A-YTHDF1-dependent manner after traumatic brain injury.

Traumatic brain injury (TBI) is a common complication of acute and severe neurosurgery. Remodeling of N6-methyladenosine (m6A) stabilization may be an attractive treatment option for neurological dysfunction after TBI. In the present study, the authors explored the epigenetic methylation of RNA-mediated NLRP3 inflammasome activation after TBI. Neurological dysfunction, histopathology, and associated molecules were examined in conditional knockout (CKO) WTAP [flox/flox, Camk2a-cre] , WTAP flox/flox , and pAAV-U6-shRNA-YTHDF1-transfected mice. Primary neurons were used in vitro to further explore the molecular mechanisms of action of WTAP/YTHDF1 following neural damage. The authors found that WTAP and m6A levels were upregulated at an early stage after TBI, and conditional deletion of WTAP in neurons did not affect neurological function but promoted functional recovery after TBI. Conditional deletion of WTAP in neurons suppressed neuroinflammation at the TBI early phase: WTAP could directly act on NLRP3 mRNA, regulate NLRP3 mRNA m6A level, and promote NLRP3 expression after neuronal injury. Further investigation found that YTH domain of YTHDF1 could directly bind to NLRP3 mRNA and regulate NLRP3 protein expression. YTHDF1 mutation or silencing improved neuronal injury, inhibited Caspase-1 activation, and decreased IL-1β levels. This effect was mediated via suppression of NLRP3 protein translation, which also reversed the stimulative effect of WTAP overexpression on NLRP3 expression and inflammation. Our results indicate that WTAP participates in neuronal damage by protein translation of NLRP3 in an m6A-YTHDF1-dependent manner after TBI and that WTAP/m6A/YTHDF1 downregulation therapeutics is a viable and promising approach for preserving neuronal function after TBI, which can provide support for targeted drug development.

Reserpine, a novel N6-methyladenosine regulator, reverses Lenvatinib resistance in hepatocellular carcinoma

BackgroundHepatocellular carcinoma (HCC) is an aggressive malignancy and a growing global health problem. Reserpine (Res), a plant-derived hypertension drug, has been reported to possess anti-tumor efficacy. However, the role and function of Res in N6-methyladenosine (m6A) regulation and Lenvatinib (Len) resistance in HCC have not been clarified. PurposeTo verify whether Res can be used as a natural small-molecule regulator of m6A to reverse Len resistance in HCC. MethodsDot blotting, Western blotting and m6A quantification were used to compare and analyze the differential expression of m6A and its methyltransferase METTL3. Western blotting, Real-Time PCR (RT-PCR), cellular thermal shift assay (CETSA) and molecular docking were used to explore the mechanism of interaction between Res and m6A. The effects of Res on the biological characteristics of Lenvatinib-resistant HCC cells were investigated through CCK-8, clone formation, and Transwell assays. Cell line-derived xenograft (CDX) and patient-derived xenograft (PDX) mouse models were used to assess the ability of Res to reverse Len resistance in vivo. MeRIP m6A sequencing, PATHWAY analysis and Western blotting were used to analyze the downstream signaling pathways and genes involved in Res-mediated reversal of Len resistance. ResultsLen resistance in HCC is related to the increased m6A level and the high expression of METTL3. Res affects the activity of METTL3 protein by binding to it, thereby downregulating the level of m6A. In vitro study showed that Res can sensitize HCC cells to the anti-tumor effects of Len treatment, including blocking proliferation, inhibiting migration, and inducing apoptosis. Len-resistant CDX and PDX models revealed that Res can reverse the resistant phenotype, with the tumor inhibition rates of 77.46 % and 62.1 %, respectively, when combined with Len treatment. Analysis of xenograft tissues showed that the combination of Res and Len down-regulates the m6A level, reduces proliferation biomarkers, and induces apoptosis, which is consistent with the in vitro data. Mechanistically, our preliminary results indicate that Res can up-regulate the SMAD3 level by down-regulating m6A in Len-resistant cells. ConclusionsReserpine, a small-molecule regulator of m6A, reverses Lenvatinib-resistant phenotypes, including proliferation, migration and anti-apoptosis, in vitro and in vivo by targeting SMAD3 and down-regulating the m6A level in HCC.

Small HBV surface antigendrives regorafenib resistance in HCC via KIAA1429-dependent m6A modification of CCR9.

A substantial body of literature, including our own, points to a connection between hepatitis B virus (HBV) infection and the development of drug resistance in hepatocellular carcinoma (HCC), particularly against sorafenib. However, the influence of HBV on resistance to regorafenib, another therapeutic agent, has been less studied. In this study, we used the GEO database (GSE87630) and clinical samples to demonstrate that C-C motif chemokine receptor 9 (CCR9) was highly expressed in HBV-related HCC and predicted poor overall survival. Its overexpression correlated with HBsAg-positive HCC patients. Both univariate and multivariable Cox regression analysis elucidated CCR9 was an independent risk factor for poor overall survival in HCC patients. Our in vitro findings further revealed that HBV structural proteins, small HBV surface antigen (SHBs), triggered an upregulation of CCR9. Functional assays showed that SHBs enhanced HCC cell proliferation, migration, and invasion, increased ABCB1 and ABCC1 expression, and promoted regorafenib resistance via CCR9. Intriguingly, overexpression of HBV plasmid and an AAV-HBV mouse model both exhibited a significant elevation in global N6-methyladenosine (m6A) levels. Further investigations revealed that SHBs elevated these m6A levels, upregulated CCR9 and stabilized CCR9 mRNA through KIAA1429-mediated m6A modification, with sites 1373 and 1496 on CCR9 mRNA being critical for modification. In conclusion, SHBs promoted HCC progression and regorafenib resistance via KIAA1429-mediatedm6A modification of CCR9. Our findings suggested that CCR9 could be a potential prognostic biomarker and a valuable molecular therapeutic target of regorafenib resistance in HBV-related HCC.

Compression Promotes the Osteogenic Differentiation of Human Periodontal Ligament Stem Cells by Regulating METTL14-mediated IGF1.

Orthodontic treatment involves the application of mechanical force to induce periodontal tissue remodeling and ultimately promote tooth movement. It is essential to study the response mechanisms of human periodontal ligament stem cells (hPDLSCs) to improve orthodontic treatment. In this study, hPDLSCs treated with compressive force were used to simulate orthodontic treatment. Cell viability and cell death were assessed using the CCK-8 assay and TUNEL staining. Alkaline phosphatase (ALP) and alizarin red staining were performed to evaluate osteogenic differentiation. The binding relationship between IGF1 and METTL14 was assessed using RIP and dual-luciferase reporter assays. The compressive force treatment promoted the viability and osteogenic differentiation of hPDLSCs. Additionally, m6A and METTL14 levels in hPDLSCs increased after compressive force treatment, whereas METTL14 knockdown decreased cell viability and inhibited the osteogenic differentiation of hPDLSCs treated with compressive force. Furthermore, the upregulation of METTL14 increased m6A levels, mRNA stability, and IGF1 expression. RIP and dual-luciferase reporter assays confirmed the interaction between METTL14 and IGF1. Furthermore, rescue experiments demonstrated that IGF1 overexpression reversed the effects of METTL14 knockdown in hPDLSCs treated with compressive force. In conclusion, this study demonstrated that compressive force promotes cell viability and osteogenic differentiation of hPDLSCs by regulating IGF1 levels mediated by METTL14.

Comprehensive Analysis of Crucial m6A-Related Differentially Expressed Genes in Psoriasis

Background: Psoriasis is a common, chronic, and multifactorial inflammatory cutaneous disorder that involves genetic and epigenetic factors. N6-methyladenosine methylation (m6A) is the most prevalent RNA modification implicated in various diseases; however, its role in psoriasis still needs to be further explored. We aimed to explore the mechanisms underlying the effects of m6A in psoriasis pathogenesis, prompting new therapeutic targets. Methods: Three psoriasis-related datasets, including GSE155702, GSE109248, and GSE142582, were collected. Differentially m6A methylated genes (DMGs) between psoriasis lesions of psoriasis patients and healthy skin controls were identified from the GSE155702 dataset, and corresponding Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed. Differentially expressed genes (DEGs) and the common DEGs between the two groups were screened from the GSE109248 and GSE142582 datasets; the expression and interactions of the m6A regulators were analyzed. The m6A levels of total RNAs and the protein expression levels of METTL3, WTAP, ALKBH5, FTO, and METTL14 in imiquimod (IMQ)-induced psoriasiform lesions were evaluated. Results: 66 significantly upregulated and 381 significantly downregulated m6A peaks were identified, corresponding to 414 genes which were particularly associated with cell and tissue development processes and cell cycle related items. 271 common DEGs were identified, associating with keratinocyte differentiation, epidermis development, cytokine-cytokine receptor interaction, and fatty acid metabolic processes. 15 crucial m6A related differentially expressed genes were obtained after the intersection of the DMGs and common DEGs, including NEU2, GALNT6, MTCL1, DOC2B, CAMK2N1, SNTB1, RNF150, CGNL1, CCDC102A, MEOX2, EEF2K, OBSCN, SLC46A2, CCDC85A, and DACH1. In addition, we found that m6A methylation and these five m6A regulators were both upregulated in psoriatic lesions. Conclusions: It revealed that psoriasis pathophysiological processes encompass m6A epigenetic alterations, and that m6A alterations may specifically influence cell proliferation and neural regulation, and closely associated with osteoarticular involvement and metabolic syndrome in psoriasis.