ALKBH5-Mediated m6A Modification of XBP1 Facilitates NSCLC Progression Through the IL-6-JAK-STAT3 Pathway.

The X-box-binding protein 1 (XBP1) is an important transcription factor during endoplasmic reticulum stress response, which was reported as an oncogene in non-small cell lung cancer (NSCLC) tumorigenesis and development. However, the regulatory mechanism of XBP1 expression in NSCLC progression was less reported. N6-methyladenosine (m6A) RNA modification is an emerging epigenetic regulatory mechanism for gene expression. This study aimed to investigate the regulatory role of the m6A modification in XBP1 expression in NSCLC. We identified XBP1 as a downstream target of ALKBH5-mediated m6A modification in A549 and PC9 cells. Knockdown of ALKBH5 increased the m6A modification and the stability of XBP1 mRNA, while overexpression of ALKBH5 had the opposite effect. Furthermore, IGF2BP3 was confirmed to be a reader of XBP1 m6A methylation and to enhance the stability of XBP1 mRNA. Additionally, IGF2BP3 knockdown significantly reversed the increase in XBP1 stability mediated by ALKBH5 depletion. In vivo and in vitro experiments demonstrated that ALKBH5/IGF2BP3 promotes the proliferation, migration, and invasion of NSCLC cells by upregulating XBP1 expression. In addition, we also showed that XBP1 promoted NSCLC cell proliferation, migration, and invasion by activating IL-6-JAK-STAT3 signaling. Our research suggested that ALKBH5-mediated m6A modification of XBP1 facilitates NSCLC progression through the IL-6-JAK-STAT3 pathway.

ALKBH5 regulates etoposide-induced cellular senescence and osteogenic differentiation in osteoporosis through mediating the m6A modification of VDAC3.

Osteoporosis, a common bone disease in older individuals, involves the progression influenced by N6-methyladenosine (m6A) modification. This study aimed to elucidate the effects of VDAC3 m6A modification on human bone mesenchymal stromal cell (BMSC) senescence and osteogenic differentiation. BMSCs were treated with etoposide to induce senescence. Senescence was assessed by ß-galactosidase staining and quantitative real-time PCR (qPCR), and osteogenic differentiation was evaluated using Western blot, alkaline phosphatase, and alizarin red S staining. VDAC3 and ALKBH5 expression were quantified by qPCR, and their interaction was assessed by RNA immunoprecipitation (RIP) and luciferase reporter assay. m6A methylation was analyzed using the Me-RIP assay. VDAC3 expression was significantly decreased in etoposide-treated BMSCs (1.00 ± 0.13 vs. 0.26 ± 0.06). VDAC3 overexpression reduced etoposide-induced senescence and promoted osteogenic differentiation. ALKBH5 overexpression inhibited VDAC3 m6A modification (1.00 ± 0.095 vs. 0.233 ± 0.177) and its stability. ALKBH5 knockdown decreased etoposide-induced senescence and promoted osteogenic differentiation, effects that were reversed by VDAC3 knockdown. YTHDF1 was identified as the m6A methylation reader, and its overexpression inhibited VDAC3 stability. We demonstrated that ALKBH5 inhibited osteogenic differentiation of etoposide-induced senescent cells through the inhibition of VDAC3 m6A modification, and YTHDF1 acted as the m6A methylation reader. These findings provide a novel theoretical basis for the treatment of osteoporosis.

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-1ß, and IL-6 were measured by enzyme-linked immunosorbent assay. Quantitative real-time polymerase chain reaction or Western blotting was performed to determine the expressions of 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.

Ouabain-mediated downregulation of ALKBH5 and IGF2BP2 inhibits the malignant progression of DLBCL.

m6A modification is a crucial epigenetic regulatory mechanism in diffuse large B-cell lymphoma (DLBCL). Low-dose cardiotonic drugs have been shown to induce apoptosis in DLBCL cells through epigenetic modulation. However, the involvement of the cardiotonic drug ouabain in the malignant progression of DLBCL remains unclear. Our study revealed that ouabain indeed contributes to the malignant progression of DLBCL through m6A modification. Through qPCR analysis, we observed a negative correlation between ouabain concentration and the expression levels of the demethylase ALKBH5 and the m6A-binding protein IGF2BP2 in DLBCL cells. Furthermore, high expression levels of ALKBH5 and IGF2BP2 were identified in both the GEO database and DLBCL patient tissue samples. Notably, elevated ALKBH5 and IGF2BP2 promoted cell proliferation both in vitro and in vivo. Inhibition of their expression rendered DLBCL cells more sensitive to ouabain treatment, resulting in significant suppression of cell proliferation, G1/S phase cell cycle arrest, and increased apoptosis. In summary, our results clarify that the demethylase ALKBH5 and the m6A-binding protein IGF2BP2 are involved in the malignant progression of DLBCL, and that the cardiotonic drug ouabain can inhibit the proliferation of DLBCL cells by inhibiting the expression of ALKBH5 and IGF2BP2, which provides new insights into the targeted treatment of DLBCL.

The role of RNA m6A demethylase ALKBH5 in the mechanisms of fibrosis.

ALKBH5 is one of the demethylases involved in the regulation of RNA m6A modification. In addition to its role in the dynamic regulation of RNA m6A modification, ALKBH5 has been found to play important roles in various tissues fibrosis processes in recent years. However, the mechanisms and effects of ALKBH5 in fibrosis have been reported inconsistently. Multiple cell types, including parenchymal cells, immune cells (neutrophils and T cells), macrophages, endothelial cells, and fibroblasts, play roles in various stages of fibrosis. Therefore, this review analyzes the mechanisms by which ALKBH5 regulates these cells, its impact on their functions, and the outcomes of fibrosis. Furthermore, this review summarizes the role of ALKBH5 in fibrotic diseases such as pulmonary fibrosis, liver fibrosis, cardiac fibrosis, and renal fibrosis, and discusses various ALKBH5 inhibitors that have been discovered to date, exploring the potential of ALKBH5 as a clinical target for fibrosis.

Unraveling molecular and clinical aspects of ALKBH5 as dual role in colorectal cancer.

OBJECTIVES: This study investigates the dual role of ALKBH5, an eraser enzyme, in colorectal cancer (CRC), focusing on how N6-methyladenosine (m6A) mutations influence CRC development and progression. METHODS: We reviewed various studies that highlighted the role of ALKBH5 in colorectal cancer (CRC). This includes the impact of ALKBH5 on tumor cell behavior including immune system interactions, invasion, and proliferation in CRC. We also looked into how ALKBH5 acts as a tumor suppressor under different conditions analyzed clinical data to assess the impact of ALKBH5 expression on outcomes in colorectal cancer patients. KEY FINDINGS: In CRC, ALKBH5 plays a dual role. In certain situations, it inhibits the progression of the tumor, but in other circumstances, it promotes tumor growth and immunosuppression. The interaction with RABA5 plays a role in the development of CRC. Having elevated levels of ALKBH5 has been associated with unfavorable patient outcomes, such as reduced survival rates and more advanced cancer stages. Various factors, including tumor differentiation, TNM stages, and carcinoembryonic antigen (CEA) levels, be linked to ALKBH5 expression. CONCLUSIONS: ALKBH5 plays a complicated and situation-specific role in colorectal cancer (CRC). Targeting ALKBH5 could result in novel therapy options that balance its tumor-promoting and tumor-fighting properties in CRC. Further research into m6A alterations and ALKBH5 could enhance CRC treatment approaches and patient outcomes.

ALKBH5-mediated m6A demethylation ameliorates extracellular matrix deposition in cutaneous pathological fibrosis.

BACKGROUND: Elevated extracellular matrix (ECM) accumulation is a major contributing factor to the pathogenesis of fibrotic diseases. Recent studies have indicated that N6-methyladenosine (m6A) RNA modification plays a pivotal role in modulating RNA stability and contribute to the initiation of various pathological conditions. Howbeit, the precise mechanism by which m6A influences ECM deposition remains unclear. METHODS: In this study, we used hypertrophic scars (HTSs) as a paradigm to investigate ECM-related diseases. We focused on the role of ALKBH5-mediated m6A demethylation within the pathological progression of HTSs and examined its correlation with clinical stages. The effects of ALKBH5 ablation on ECM components were studied both in vivo and in vitro. Downstream targets of ALKBH5, along with their underlying mechanisms, were identified using integrated high-throughput analysis, RNA-binding protein immunoprecipitation and RNA pull-down assays. Furthermore, the therapeutic potential of exogenous ALKBH5 overexpression was evaluated in fibrotic scar models. RESULTS: ALKBH5 was decreased in fibroblasts derived from HTS lesions and was negatively correlated with their clinical stages. Importantly, ablation of ALKBH5 promoted the expression of COL3A1, COL1A1, and ELN, leading to pathological deposition and reconstruction of the ECM both in vivo and in vitro. From a therapeutic perspective, the exogenous overexpression of ALKBH5 significantly inhibited abnormal collagen deposition in fibrotic scar models. As determined by integrated high-throughput analysis, key ECM components including COL3A1, COL1A1, and ELN are direct downstream targets of ALKBH5. By means of its mechanism, ALKBH5 inhibits the expression of COL3A1, COL1A1, and ELN by removing m6A from mRNAs, thereby decreasing their stability in a YTHDF1-dependent manner. CONCLUSIONS: Our study identified ALKBH5 as an endogenous suppressor of pathological ECM deposition, contributing to the development of a reprogrammed m6A-targeted therapy for HTSs.

ALKBH5 deficiency attenuates oxygen-glucose deprivation-induced injury in mouse brain microvascular endothelial cells in an m6A dependent manner.

Structural and functional alterations in brain microvascular endothelial cells (BMECs) caused by oxygen-glucose deprivation (OGD) are involved in the pathogenesis of various brain disorders. AlkB homolog 5 (ALKBH5) is a primary m6A demethylase that regulates various cell processes, but its distinct roles in BMEC function remain to be clarified. In the present study, in mouse middle cerebral artery occlusion (MCAO) model, knockout of ALKBH5 reduced neurological deficits, infarct volumes and tissue apoptosis caused by ischemia/reperfusion injury. Evans blue leakage and decreased expression of the tight junction protein ZO-1 and Occludin were also attenuated by ALKBH5 knockout. During the exploration of the underlying mechanisms of the role of ALKBH5 in BMECs, we found that the expression of ALKBH5 was induced at both the mRNA and protein levels by hypoxia; however, its protein stability was impaired by OGD treatment. Knockdown of ALKBH5 expression increased total m6A levels and alleviated OGD-induced BMEC injury. At the same time, the selective ALKBH5 inhibitor Cpd 20m also exhibited a protective effect on cell injury. In contrast, overexpression of ALKBH5 increased the sensitivity of BMECs to OGD. Interestingly, the m6A sequencing data revealed that knockdown of ALKBH5altered the expression of many genes via m6A upregulation. The gene expression alterations were verified by real-time PCR. Taken together, our results suggest that ALKBH5, as well as its target genes, plays important roles in the regulation of brain microvascular endothelial cell function through its RNA demethylase activity.

The RNA Demethylases ALKBH5 and FTO Regulate the Translation of ATF4 mRNA in Sorafenib-Treated Hepatocarcinoma Cells.

Translation is one of the main gene expression steps targeted by cellular stress, commonly referred to as translational stress, which includes treatment with anticancer drugs. While translational stress blocks the translation initiation of bulk mRNAs, it nonetheless activates the translation of specific mRNAs known as short upstream open reading frames (uORFs)-mRNAs. Among these, the ATF4 mRNA encodes a transcription factor that reprograms gene expression in cells responding to various stresses. Although the stress-induced translation of the ATF4 mRNA relies on the presence of uORFs (upstream to the main ATF4 ORF), the mechanisms mediating this effect, particularly during chemoresistance, remain elusive. Here, we report that ALKBH5 (AlkB Homolog 5) and FTO (FTO: Fat mass and obesity-associated protein), the two RNA demethylating enzymes, promote the translation of ATF4 mRNA in a transformed liver cell line (Hep3B) treated with the chemotherapeutic drug sorafenib. Using the in vitro luciferase reporter translational assay, we found that depletion of both enzymes reduced the translation of the reporter ATF4 mRNA upon drug treatment. Consistently, depletion of either protein abrogates the loading of the ATF3 mRNA into translating ribosomes as assessed by polyribosome assays coupled to RT-qPCR. Collectively, these results indicate that the ALKBH5 and FTO-mediated translation of the ATF4 mRNA is regulated at its initiation step. Using in vitro methylation assays, we found that ALKBH5 is required for the inhibition of the methylation of a reporter ATF4 mRNA at a conserved adenosine (A235) site located at its uORF2, suggesting that ALKBH5-mediated translation of ATF4 mRNA involves demethylation of its A235. Preventing methylation of A235 by introducing an A/G mutation into an ATF4 mRNA reporter renders its translation insensitive to ALKBH5 depletion, supporting the role of ALKBH5 demethylation activity in translation. Finally, targeting either ALKBH5 or FTO sensitizes Hep3B to sorafenib-induced cell death, contributing to their resistance. In summary, our data show that ALKBH5 and FTO are novel factors that promote resistance to sorafenib treatment, in part by mediating the translation of ATF4 mRNA.

Insights into the m6A demethylases FTO and ALKBH5 : structural, biological function, and inhibitor development.

N6-methyladenosine (m6A) is dynamically regulated by methyltransferases (termed "writers") and demethylases (referred to as "erasers"), facilitating a reversible modulation. Changes in m6A levels significantly influence cellular functions, such as RNA export from the nucleus, mRNA metabolism, protein synthesis, and RNA splicing. They are intricately associated with a spectrum of pathologies. Moreover, dysregulation of m6A modulation has emerged as a promising therapeutic target across many diseases. m6A plays a pivotal role in controlling vital downstream molecules and critical biological pathways, contributing to the pathogenesis and evolution of numerous conditions. This review provides an overview of m6A demethylases, explicitly detailing the structural and functional characteristics of FTO and ALKBH5. Additionally, we explore their distinct involvement in various diseases, examine factors regulating their expression, and discuss the progress in inhibitor development.

ALKBH5-Mediated m6A Modification Drives Apoptosis in Renal Tubular Epithelial Cells by Negatively Regulating MUC1.

Dysregulation of renal tubular epithelial cell (RTEC) apoptosis is one of the critical steps underlying the occurrence and development of nephrolithiasis. Although N6-methyladenosine (m6A) modification has been extensively studied and associated with various pathologic processes, research on its specific role in RTEC injury and apoptosis remains limited. In this study, we found that overexpression of ALKBH5 reduced the level of m6A modification in RTEC cells and notably promoted RTEC apoptosis. Further mechanism studies revealed that ALKBH5 mainly  decreased the m6A level on the mRNA of  Mucin 1 (MUC1) gene in RTECs. Moreover, ALKBH5  impaired the stability of MUC1 mRNA in RTECs, leading to  attenuated expression of MUC1. Finally, we determined that the ALKBH5-MUC1 axis primarily facilitated RTEC apoptosis by regulating the PI3K/Akt signaling pathway. This study revealed the critical role of the ALKBH5-MUC1-PI3K/Akt regulatory system in RTEC apoptosis and provided new therapeutic targets for treating nephrolithiasis.

Complicated role of ALKBH5 in gastrointestinal cancer: an updated review.

Gastrointestinal cancer is the most common malignancy in humans, often accompanied by poor prognosis. N6-methyladenosine (m6A) modification is widely present in eukaryotic cells as the most abundant RNA modification. It plays a crucial role in RNA splicing and processing, nuclear export, translation, and stability. Human AlkB homolog 5 (ALKBH5) is a type of RNA demethylase exhibiting abnormal expression in various gastrointestinal cancers.It is closely related to the tumorigenesis, proliferation, migration, and other biological functions of gastrointestinal cancer. However, recent studies indicated that the role and mechanism of ALKBH5 in gastrointestinal cancer are complicated and even controversial. Thus, this review summarizes recent advances in elucidating the role of ALKBH5 as a tumor suppressor or promoter in gastrointestinal cancer. It examines the biological functions of ALKBH5 and its potential as a therapeutic target, providing new perspectives and insights for gastrointestinal cancer research.

N6-methyladenosine demethylase ALKBH5 homologous protein protects against cerebral I/R injury though suppressing SNHG3-mediated neural PANoptosis: Involvement of m6A-related macromolecules in the diseases of nervous system.

In order to address this gap in knowledge, the present study utilized both in vivo and in vitro models to investigate the role of the m6A demethylase ALKBH5 in protecting against cerebral I/R injury by inhibiting PANoptosis (Pytoptosis, Ppoptosis, and Necroptosis) in an m6A-dependent manner. They observed that ALKBH5, the predominant m6A demethylase, was downregulated in these models, while SNHG3 and PANoptosis-related proteins (ZBP1, AIM2, Cappase-3, Caspase-8, cleaved Caspase-1, GSDMD-N, and p-MLKL) were elevated. Additionally, both ALKBH5 overexpression and SNHG3-deficiency were found to ameliorate PANoptosis and injury induced by OGD/reperfusion and OGD/RX in both mice tissues and astrocyte cells. Further experiments demonstrated that ALKBH5 induced m6A-demethylation in SNHG3, leading to its degradation. Low expression of SNHG3, on the other hand, prevented the formation of the SNHG3-ELAVL1-ZBP1/AIM2 complex, which in turn destabilized ZBP1 and AIM2 mRNA, resulting in the downregulation of these PANoptosis-related genes. Ultimately, the rescue experiments provided evidence that ALKBH5 protected against PANoptosis in cerebral I/R injury models through the inhibition of SNHG3.This study sheds light on the intricate molecular mechanisms involved in the pathogenesis of cerebral I/R injury and highlights the potential of m6A-related genes as therapeutic targets in this condition.

E3 ubiquitin ligase RNF180 mediates the ALKBH5/SMARCA5 axis to promote colon inflammation and Th17/Treg imbalance in ulcerative colitis mice.

SMARCA5, a protein in the SWI/SNF family, has been previously implicated in the development of ulcerative colitis (UC) through methylation. However, the specific molecular mechanisms by which SMARCA5 contributes to colonic inflammation and the imbalance between Th17 and Treg cells remain unclear. This study was designed to explore these molecular mechanisms. A UC mouse model was established using dextran sulfate sodium induction, followed by measurements of mouse weight, disease activity index (DAI) score, colon length, pathological changes in the colon, and FITC-dextran concentration. The levels of IL-17a, IFN-γ, IL-6, TNF-α, TGF-ß, and IL-10 were measured, along with the protein expression of ZO-1 and Occludin. Flow cytometry was used to assess the presence of IL-17 + CD4 + (Th17 +) cells and FOXP3 + CD25 + CD4 + (Treg +) cells in the spleen and mesenteric lymph nodes of UC mice. We observed that SMARCA5 and RNF180 were increased, while ALKBH5 was downregulated in UC mouse colon tissue. SMARCA5 or RNF180 knockdown or ALKBH5 overexpression ameliorated the colon inflammation and Th17/Treg cell imbalance in UC mice, shown by increased body weight, colon length, FOXP3 + CD25 + CD4 + T cells, and the levels of ZO-1, Occludin, TGF-ß, IL-10, and FOXP3. It decreased DAI scores, IL-17 + CD4 + T cells, and levels of IL-17a, IFN-γ, IL-6, TNF-α, and ROR-γt. ALKBH5 inhibited SMARCA5 expression via m6A modification, while RNF180 reduced ALKBH5 expression via ubiquitination. Our findings indicate that RNF180 aggravated the colon inflammation and Th17/Treg cell imbalance in UC mice by regulating the ALKBH5/SMARCA5 axis.

The methyltransferase METTL3 regulates endothelial cell proliferation and inflammation via m6A RNA methylation-mediated TRAF1 expression.

The imbalance of vascular endothelial cell homeostasis is the key mechanism for the progression of many vascular diseases. RNA modification, particularly N6-Methyladenosine (m6A), plays important function in numerous biological processes. Nevertheless, the regulatory function of m6A RNA methylation in endothelial dysfunction remains insufficiently characterized. In this study, we established that the m6A methyltransferase METTL3 is critical for regulating endothelial function. Functionally, depletion of METTL3 results in decreased endothelial cells proliferation, survival and inflammatory response. Conversely, overexpression of METTL3 elicited the opposite effects. Mechanistically, MeRIP-seq identified that METTL3 catalyzed m6A modification of TRAF1 mRNA and enhanced TRAF1 translation, thereby up-regulation of TRAF1 protein. Over-expression of TRAF1 successfully rescued the inhibition of proliferation and adhesion of endothelial cells due to METTL3 knockdown. Additionally, m6A methylation-mediated TRAF1 expression can be reversed by the demethylase ALKBH5. Knockdown of ALKBH5 upregulated the level of m6A and protein level of TRAF1, and also increased endothelial cells adhesion and inflammatory response. Collectively, our findings suggest that METTL3 regulates vascular endothelium homeostasis through TRAF1 m6A modification, suggesting that targeting the METTL3-m6A-TRAF1 axis may hold therapeutic potential for patients with vascular diseases.

ALKBH5 gene polymorphisms and risk of neuroblastoma in Chinese children from Jiangsu Province.

Background: Neuroblastoma is one of the most common extracranial malignant solid tumors in children. AlkB homolog 5 (ALKBH5) is an RNA N6-methyladenosine (m6A) demethylase that plays a critical role in tumorigenesis and development. We assessed the association between single nucleotide polymorphisms (SNPs) in ALKBH5 and the risk of neuroblastoma in a case-control study including 402 patients and 473 non-cancer controls. Methods: Genotyping was determined by the TaqMan method. The association between ALKBH5 polymorphisms (rs1378602 and rs8400) and the risk of neuroblastoma was evaluated using the odds ratio (OR) and 95% confidence interval (CI). Results: We found no strong association of ALKBH5 rs1378602 and rs8400 with neuroblastoma risk. Further stratification analysis by age, sex, primary site, and clinical stage showed that the rs1378602 AG/AA genotype was associated with a lower risk of neuroblastoma in males (adjusted OR = 0.58, 95% CI = 0.35-0.97, p = 0.036) and children with retroperitoneal neuroblastoma (adjusted OR = 0.58, 95% CI = 0.34-0.98, p = 0.040). Conclusions: ALKBH5 SNPs do not seem to be associated with neuroblastoma risk. More studies are required to confirm this negative result and reveal the relationship between gene polymorphisms of the m6A modifier ALKBH5 and neuroblastoma.

m6A mRNA methylation-mediated MAPK signaling modulates the nasal mucosa inflammatory response in allergic rhinitis.

Background: Allergic rhinitis (AR) is a complex disease in which gene-environment interactions contribute to its pathogenesis. Epigenetic modifications, such as N6-methyladenosine (m6A) modification of mRNA, play important roles in regulating gene expression in multiple physiological and pathological processes. However, the function of m6A modification in AR and the inflammatory response is poorly understood. Methods: We used the ovalbumin (OVA) and aluminum hydroxide to induce an AR mouse model. Nasal symptoms, histopathology, and serum cytokines were examined. We performed combined m6A and RNA sequencing to analyze changes in m6A modification profiles. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and methylated RNA immunoprecipitation sequencing qPCR (MeRIP-qPCR) were used to verify differential methylation of mRNAs and the m6A methylation level. Knockdown or inhibition of Alkbh5 in nasal mucosa of mice was mediated by lentiviral infection or IOX1 treatment. Results: We showed that m6A was enriched in a group of genes involved in MAPK signaling pathway. Moreover, we identified a MAPK pathway involving Map3k8, Erk2, and Nfκb1 that may play a role in the disrupted inflammatory response associated with nasal inflammation. The m6A eraser, Alkbh5, was highly expressed in the nasal mucosa of AR model mice. Furthermore, knockdown of Alkbh5 expression by lentiviral infection resulted in high MAPK pathway activity and a significant nasal mucosa inflammatory response. Our findings indicate that ALKBH5-mediated m6A dysregulation likely contributes to a nasal inflammatory response via the MAPK pathway. Conclusion: Together, our data show that m6A dysregulation mediated by ALKBH5, is likely to contribute to inflammation of the nasal mucosa via the MAPK signaling pathway, suggesting that ALKBH5 is a potential biomarker for AR treatment.

Inhibition of inflammation and apoptosis through the cyclic GMP-AMP synthase-stimulator of interferon genes pathway by stress granules after ALKBH5 demethylase activation during diabetic myocardial ischaemia-reperfusion injury.

AIM: Post-transcriptional modifications and their specific mechanisms are the focus of research on the regulation of myocardial damage. Stress granules (SGs) can inhibit the inflammatory response by inhibiting the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway. This study investigated whether alkylation repair homologue protein 5 (ALKBH5) could affect myocardial inflammation and apoptosis during diabetic myocardial ischaemia-reperfusion injury (IRI) through the cGAS-STING pathway via SGs. METHODS: A diabetes ischaemia-reperfusion rat model and a high glucose hypoxia/reoxygenation cell model were established. Adeno-associated virus (AAV) and lentivirus (LV) were used to overexpress ALKBH5, while the SG agonist arsenite (Ars) and the SG inhibitor anisomycin were used as interventions. Then, the levels of apoptosis and related indicators in the cell and rat models were measured. RESULTS: In the in vivo experiment, compared with the normal sham group, the degree of myocardial tissue damage, creatine kinase-MB and cardiac troponin I in serum, and myocardial apoptosis, the infarcted area of myocardium, and the level of B-cell lymphoma 2 associated X protein, cGAS-STING pathway and inflammatory factors in the diabetes ischaemia-reperfusion group were significantly increased. However, the expression of SGs and the levels of ALKBH5, rat sarcoma-GTPase-activating protein-binding protein 1, T-cell intracellular antigen-1 and Bcl2 were significantly decreased. After AAV-ALKBH5 intervention, the degree of myocardial tissue damage, degree of myocardial apoptosis, and extent of myocardial infarction in myocardial tissue were significantly decreased. In the in vitro experiment, compared with those in the normal control group, the levels of lactate dehydrogenase, inflammation and apoptosis were significantly greater, and cell viability and the levels of ALKBH5 and SGs were decreased in the high glucose and hypoxia/reoxygenation groups. In the high glucose hypoxia/reoxygenation cell model, the degree of cell damage, inflammation, and apoptosis was greater than those in the high glucose and hypoxia/reoxygenation models, and the levels of ALKBH5 and SGs were further decreased. LV-ALKBH5 and Ars alleviated the degree of cell damage and inhibited inflammation and cell apoptosis. The inhibition of SGs could partly reverse the protective effect of LV-ALKBH5. The cGAS agonist G140 antagonized the inhibitory effects of the SG agonist Ars on cardiomyocyte apoptosis, inflammation and the cGAS-STING pathway. CONCLUSION: Both ALKBH5 and SGs inhibited myocardial inflammation and apoptosis during diabetic myocardial ischaemia-reperfusion. Mechanistically, ALKBH5 might inhibit the apoptosis of cardiomyocytes by promoting the expression of SGs through the cGAS-STING pathway.

Identification of differential m6A RNA methylomes and ALKBH5 as a potential prevention target in the developmental neurotoxicity induced by multiple sevoflurane exposures.

Sevoflurane, as a commonly used inhaled anesthetic for pediatric patients, has been reported that multiple sevoflurane exposures are associated with a greater risk of developing neurocognitive disorder. N6-Methyladenosine (m6A), as the most common mRNA modification in eukaryotes, has emerged as a crucial regulator of brain function in processes involving synaptic plasticity, learning and memory, and neurodevelopment. Nevertheless, the relevance of m6A RNA methylation in the multiple sevoflurane exposure-induced developmental neurotoxicity remains mostly elusive. Herein, we evaluated the genome-wide m6A RNA modification and gene expression in hippocampus of mice that received with multiple sevoflurane exposures using m6A-sequencing (m6A-seq) and RNA-sequencing (RNA-seq). We discovered 19 genes with differences in the m6A methylated modification and differential expression in the hippocampus. Among these genes, we determined that a total of nine differential expressed genes may be closely associated with the occurrence of developmental neurotoxicity induced by multiple sevoflurane exposures. We further found that the alkB homolog 5 (ALKBH5), but not methyltransferase-like 3 (METTL3) and Wilms tumor 1-associated protein (WTAP), were increased in the hippocampus of mice that received with multiple sevoflurane exposures. And the IOX1, as an inhibitor of ALKBH5, significantly improved the learning and memory defects and reduced neuronal damage in the hippocampus of mice induced by multiple sevoflurane exposures. The current study revealed the role of m6A methylated modification and m6A-related regulators in sevoflurane-induced cognitive impairment, which might provide a novel insight into identifying biomarkers and therapeutic strategies for inhaled anesthetic-induced developmental neurotoxicity.