LncRNA-PCat19 acts as a ceRNA of miR-378a-3p to facilitate microglia activation and accelerate chronic neuropathic pain in rats by promoting KDM3A-mediated BDNF demethylation.

The pathogenesis of neuropathic pain (NP) is complex, and there are various pathological processes. Previous studies have suggested that lncRNA PCAT19 is abnormally expressed in NP conduction and affects the occurrence and development of pain. The aim of this study is to analyze the role and mechanism of PCAT19 in NP induced by chronic compressive nerve injury (CCI) in mice. In this study, C57BL/6 mice were applied to establish the CCI model. sh-PCAT19 was intrathecally injected once a day for 5 consecutive days from the second day after surgery. We discovered that PCat19 level was gradually up-regulated with the passage of modeling time. Downregulation of Iba-1-positive expression, M1/M2 ratio of microglia, and pro-inflammatory factors in the spinal cords of CCI-mice after PCat19 knock-downed was observed. Mechanically, the expression of miR-378a-3p was negatively correlated with KDM3A and PCat19. Deletion of KDM3A prevented H3K9me2 demethylation of BDNF promoter and suppressed BDNF expression. Further, KDM3A promotes CCI-induced neuroinflammation and microglia activation by mediating Brain-derived neurotrophic factor (BDNF) demethylation. Together, the results suggest that PCat19 may be involved in the development of NP and that PCat19 shRNA injection can attenuate microglia-induced neuroinflammation by blocking KDM3A-mediated demethylation of BDNF and BDNF release.

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

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

FTO promotes gastric cancer progression by modulating MAP4K4 expression via demethylation in an m6A-dependent manner.

Gastric cancer (GC) is a serious malignant tumour with a high mortality rate and a poor prognosis. Recently, emerging evidence has suggested that N6-methyladenosine (m6A) modification plays a crucial regulatory role in cancer progression. However, the exact role of m6A regulatory factors FTO in GC is unclear. First, the expression of m6A methylation-related regulatory factors in clinical samples and the clinical data of the corresponding patients were obtained from The Cancer Genome Atlas (TCGA-STAD) dataset, and correlation analysis between FTO expression and patient clinicopathological parameters was subsequently performed. qRT-PCR, immunohistochemistry (IHC) and western blotting (WB) were used to verify FTO expression in GC. CCK-8, EdU, flow cytometry and transwell assays were used to evaluate the effect of FTO on the behaviour of GC cells. Transcriptome sequencing and RNA immunoprecipitation analysis were used to explore the potential regulatory mechanisms mediated by FTO. FTO was highly expressed in GC tissues and cells, and high expression of FTO predicted a worse prognosis than low expression. Functionally, overexpression of FTO promoted the proliferation, migration and invasion of GC cells but inhibited cell apoptosis. Mechanistically, we found that FTO is upregulated in GC and promotes GC progression by modulating the expression of MAP4K4. Taken together, our findings provide new insights into the effects of FTO-mediated m6A demethylation and could lead to the development of new strategies for GC monitoring and aggressive treatment.

Integration of Demethylation-Activated DNAzyme with a Single Quantum Dot Nanosensor for Sensitive Detection of O6-Methylguanine DNA Methyltransferase in Breast Tissues.

O6-Methylguanine-DNA-methyltransferase (MGMT) is a demethylation protein that dynamically regulates the O6-methylguanine modification (O6 MeG), and dysregulated MGMT is implicated in various malignant tumors. Herein, we integrate demethylation-activated DNAzyme with a single quantum dot nanosensor to sensitively detect MGMT in breast tissues. The presence of MGMT induces the demethylation of the O6 MeG-caged DNAzyme and the restoration of catalytic activity. The activated DNAzyme then specifically cleaves the ribonucleic acid site of hairpin DNA to expose toehold sequences. The liberated toehold sequence may act as a primer to trigger a cyclic exponential amplification reaction for the generation of enormous signal strands that bind with the Cy5/biotin-labeled probes to form sandwich hybrids. The assembly of sandwich hybrids onto 605QD obtains 605QD-dsDNA-Cy5 nanostructures, inducing efficient FRET between the 605QD donor and Cy5 acceptor. Notably, the introduction of a mismatched base in hairpin DNA can greatly minimize the background and improve the signal-to-noise ratio. This nanosensor achieves a dynamic range of 1.0 × 10-8 to 0.1 ng/µL and a detection limit of 155.78 aM, and it can screen MGMT inhibitors and monitor cellular MGMT activity with single-cell sensitivity. Moreover, it can distinguish the MGMT level in tissues of breast cancer patients and healthy persons, holding great potential in clinical diagnostics and epigenetic research studies.

FSH induces EMT in ovarian cancer via ALKBH5-regulated Snail m6A demethylation.

Background: The therapeutic benefits of targeting follicle-stimulating hormone (FSH) receptor in treatment of ovarian cancer are significant, whereas the role of FSH in ovarian cancer progresses and the underlying mechanism remains to be developed. Methods: Tissue microarray of human ovarian cancer, tumor xenograft mouse model, and in vitro cell culture were used to investigate the role of FSH in ovarian carcinogenesis. siRNA, lentivirus and inhibitors were used to trigger the inactivation of genes, and plasmids were used to increase transcription of genes. Specifically, pathological characteristic was assessed by histology and immunohistochemistry (IHC), while signaling pathway was studied using western blot, quantitative RT-PCR, and immunofluorescence. Results: Histology and IHC of human normal ovarian and tumor tissue confirmed the association between FSH and Snail in ovarian cancer metastasis. Moreover, in epithelial ovarian cancer cells and xenograft mice, FSH was showed to promote epithelial mesenchymal transition (EMT) progress and metastasis of ovarian cancer via prolonging the half-life of Snail mRNA in a N6-methyladenine methylation (m6A) dependent manner, which was mechanistically through the CREB/ALKBH5 signaling pathway. Conclusions: These findings indicated that FSH induces EMT progression and ovarian cancer metastasis via CREB/ALKBH5/Snail pathway. Thus, this study provided new insight into the therapeutic strategy of ovarian cancer patients with high level of FSH.

FTO-mediated m6A mRNA demethylation aggravates renal fibrosis by targeting RUNX1 and further enhancing PI3K/AKT pathway.

Chronic kidney disease (CKD) is a global health burden, with ineffective therapies leading to increasing morbidity and mortality. Renal interstitial fibrosis is a common pathway in advanced CKD, resulting in kidney function and structure deterioration. In this study, we investigate the role of FTO-mediated N6-methyladenosine (m6A) and its downstream targets in the pathogenesis of renal fibrosis. M6A modification, a prevalent mRNA internal modification, has been implicated in various organ fibrosis processes. We use a mouse model of unilateral ureteral obstruction (UUO) as an in vivo model and treated tubular epithelial cells (TECs) with transforming growth factor (TGF)-ß1 as in vitro models. Our findings revealed increased FTO expression in UUO mouse model and TGF-ß1-treated TECs. By modulating FTO expression through FTO heterozygous mutation mice (FTO+/- ) in vivo and small interfering RNA (siRNA) in vitro, we observed attenuation of UUO and TGF-ß1-induced epithelial-mesenchymal transition (EMT), as evidenced by decreased fibronectin and N-cadherin accumulation and increased E-cadherin levels. Silencing FTO significantly improved UUO and TGF-ß1-induced inflammation, apoptosis, and inhibition of autophagy. Further transcriptomic assays identified RUNX1 as a downstream candidate target of FTO. Inhibiting FTO was shown to counteract UUO/TGF-ß1-induced RUNX1 elevation in vivo and in vitro. We demonstrated that FTO signaling contributes to the elevation of RUNX1 by demethylating RUNX1 mRNA and improving its stability. Finally, we revealed that the PI3K/AKT pathway may be activated downstream of the FTO/RUNX1 axis in the pathogenesis of renal fibrosis. In conclusion, identifying small-molecule compounds that target this axis could offer promising therapeutic strategies for treating renal fibrosis.

ALKBH5 facilitates the progression of skin cutaneous melanoma via mediating ABCA1 demethylation and modulating autophagy in an m6A-dependent manner.

Background: N6-methyladenosine (m6A) is the most common and abundant mRNA modification, playing an essential role in biological processes and tumor development. However, the role of m6A methylation in skin cutaneous melanoma (SKCM) is not yet clear. This study analyzed the expression of m6A-related functional genes in SKCM and aimed to explore the key demethylase ALKBH5 mediated m6A modification and its potential mechanism in human SKCM. Methods: Based on public databases, the m6A-related gene expression landscape in SKCM was portrayed. MeRIP-Seq and RNA-Seq were used to recognize the downstream target of ALKBH5. In vivo and in vitro functional phenotype and rescue functional experiments were performed to explore the mechanism of the ALKBH5-m6A-ABCA1 axis in SKCM. Results: We found ALKBH5 upregulated in SKCM, associated with poor prognosis. ALKBH5 can promote melanoma cell proliferation, colony formation, migration, and invasion and inhibit autophagy in vitro, facilitating tumor growth and metastasis in vivo. We identified ABCA1, a membrane protein that assists cholesterol efflux, as a downstream target of ALKBH5-mediated m6A demethylation. Finally, our data demonstrated that ALKBH5 promoted SKCM via mediating ABCA1 downregulation by reducing ABCA1 mRNA stability in an m6A-dependent manner. Conclusion: Our findings exhibited the functional value of the key demethylase ALKBH5 mediated m6A modification in the progression of SKCM, suggesting the ALKBH5-m6A-ABCA1 axis as a potential therapeutic target in SKCM.

The antagonistic effect of FTO on METTL14 promotes AKT3 m6A demethylation and the progression of esophageal cancer.

BACKGROUND: As the most abundant modification in eukaryotic messenger RNAs (mRNAs), N6-methyladenosine (m6A) plays vital roles in many biological processes. METHODS: Methylated RNA immunoprecipitation sequencing (MeRIP-seq) and transcriptomic RNA sequencing (RNA-seq) were used to screen for m6A targets in esophageal cancer cells and patients. The role of m6A RNA methylase in esophageal cancer was also analyzed using bioinformatics. In vitro and in vivo experiments were used to analyze gene expression and function. CCK-8, colony formation, cell apoptosis and immunofluorescence staining assays were performed to evaluate the proliferation, migration and invasion of esophageal cancer cells, respectively. Western blot analysis, RNA stability, RIP and luciferase reporter assays were performed to elucidate the underlying mechanism involved. RESULTS: We found that the m6A demethylase FTO was significantly upregulated in esophageal cancer cell lines and patient tissues. In vivo and in vitro assays demonstrated that FTO was involved in the proliferation and apoptosis of esophageal cancer cells. Moreover, we found that the m6A methyltransferase METTL14 negatively regulates FTO function in esophageal cancer progression. FTO alone is not related to the prognosis of esophageal cancer, and its function is antagonized by METTL14. By using transcriptome-wide m6A-seq and RNA-seq assays, we revealed that AKT3 is a downstream target of FTO and acts in concert to regulate the tumorigenesis and metastasis of esophageal cancer. Taken together, these findings provide insight into m6A-mediated tumorigenesis in esophageal cancer and could lead to the design of new therapeutic strategies.

Targeted demethylation and activation of NLRC5 augment cancer immunogenicity through MHC class I.

Impaired expression of MHC (major histocompatibility complex) class I in cancers constitutes a major mechanism of immune evasion. It has been well documented that the low level of MHC class I is associated with poor prognosis and resistance to checkpoint blockade therapies. However, there is lmited approaches to specifically induce MHC class I to date. Here, we show an approach for robust and specific induction of MHC class I by targeting an MHC class I transactivator (CITA)/NLRC5, using a CRISPR/Cas9-based gene-specific system, designated TRED-I (Targeted reactivation and demethylation for MHC-I). The TRED-I system specifically recruits a demethylating enzyme and transcriptional activators on the NLRC5 promoter, driving increased MHC class I antigen presentation and accelerated CD8+ T cell activation. Introduction of the TRED-I system in an animal cancer model exhibited tumor-suppressive effects accompanied with increased infiltration and activation of CD8+ T cells. Moreover, this approach boosted the efficacy of checkpoint blockade therapy using anti-PD1 (programmed cell death protein) antibody. Therefore, targeting NLRC5 by this strategy provides an attractive therapeutic approach for cancer.

DNA polymerase beta connects tumorigenicity with the circadian clock in liver cancer through the epigenetic demethylation of Per1.

The circadian-controlled DNA repair exhibits a strong diurnal rhythm. Disruption in circadian clock and DNA repair is closely linked with hepatocellular carcinoma (HCC) progression, but the mechanism remains unknown. Here, we show that polymerase beta (POLB), a critical enzyme in the DNA base excision repair pathway, is rhythmically expressed at the translational level in mouse livers. Hepatic POLB dysfunction dampens clock homeostasis, whereas retards HCC progression, by mediating the methylation of the 4th CpG island on the 5'UTR of clock gene Per1. Clinically, POLB is overexpressed in human HCC samples and positively associated with poor prognosis. Furthermore, the hepatic rhythmicity of POLB protein expression is orchestrated by Calreticulin (CALR). Our findings provide important insights into the molecular mechanism underlying the synergy between clock and food signals on the POLB-driven BER system and reveal new clock-dependent carcinogenetic effects of POLB. Therefore, chronobiological modulation of POLB may help to promote precise interventions for HCC.

Auto-suppression of Tet dioxygenases protects the mouse oocyte genome from oxidative demethylation.

DNA cytosine methylation plays a vital role in repressing retrotransposons, and such derepression is linked with developmental failure, tumorigenesis and aging. DNA methylation patterns are formed by precisely regulated actions of DNA methylation writers (DNA methyltransferases) and erasers (TET, ten-eleven translocation dioxygenases). However, the mechanisms underlying target-specific oxidation of 5mC by TET dioxygenases remain largely unexplored. Here we show that a large low-complexity domain (LCD), located in the catalytic part of Tet enzymes, negatively regulates the dioxygenase activity. Recombinant Tet3 lacking LCD is shown to be hyperactive in converting 5mC into oxidized species in vitro. Endogenous expression of the hyperactive Tet3 mutant in mouse oocytes results in genome-wide 5mC oxidation. Notably, the occurrence of aberrant 5mC oxidation correlates with a consequent loss of the repressive histone mark H3K9me3 at ERVK retrotransposons. The erosion of both 5mC and H3K9me3 causes ERVK derepression along with upregulation of their neighboring genes, potentially leading to the impairment of oocyte development. These findings suggest that Tet dioxygenases use an intrinsic auto-regulatory mechanism to tightly regulate their enzymatic activity, thus achieving spatiotemporal specificity of methylome reprogramming, and highlight the importance of methylome integrity for development.

Targeted m6A demethylation of ITGA6 mRNA by a multisite dCasRx-m6A editor inhibits bladder cancer development.

INTRODUCTION: N6-methyladenosine (m6A) modification contributes to the pathogenesis and development of various cancers, including bladder cancer (BCa). In particular, integrin α6 (ITGA6) promotes BCa progression by cooperatively regulating multisite m6A modification. However, the therapeutic effect of targeting ITGA6 multisite m6A modifications in BCa remains unknown. OBJECTIVES: We aim to develop a multisite dCasRx- m6A editor for assessing the effects of the multisite dCasRx-m6A editor targeted m6A demethylation of ITGA6 mRNA in BC growth and progression. METHODS: The multisite dCasRx- m6A editor was generated by cloning. m6A-methylated RNA immunoprecipitation (meRIP), luciferase reporter, a single-base T3 ligase-based qPCR-amplification, Polysome profiling and meRIP-seq experiments were performed to determine the targeting specificity of the multisite dCasRx-m6A editor. We performed cell phenotype analysis and used in vivo mouse xenograft models to assess the effects of the multisite dCasRx-m6A editor in BC growth and progression. RESULTS: We designed a targeted ITGA6 multi-locus guide (g)RNA and established a bidirectional deactivated RfxCas13d (dCasRx)-based m6A-editing platform, comprising a nucleus-localized dCasRx fused with the catalytic domains of methyltransferase-like 3 (METTL3-CD) or α-ketoglutarate-dependent dioxygenase alkB homolog 5 (ALKBH5-CD), to simultaneously manipulate the methylation of ITGA6 mRNA at four m6A sites. The results confirmed the dCasRx-m6A editor modified m6A at multiple sites in ITGA6 mRNA, with low off-target effects. Moreover, targeted m6A demethylation of ITGA6 mRNA by the multisite dCasRx-m6A editor significantly reduced BCa cell proliferation and migration in vitro and in vivo. Furthermore, the dCasRx-ALKBH5-CD and ITGA6 multi-site gRNA delivered to 5-week-old BALB/cJNju-Foxn1nu/Nju nude mice via adeno-associated viral vectors significantly inhibited BCa cell growth. CONCLUSION: Our study proposes a novel therapeutic tool for the treatment of BC by applying the multisite dCasRx-m6A editor while highlighting its potential efficacy for treating other diseases associated with abnormal m6A modifications.

LncRNA GAS5 regulated by FTO-mediated m6A demethylation promotes autophagic cell death in NSCLC by targeting UPF1/BRD4 axis.

Long non-coding RNA (lncRNA) growth arrest-specific transcript 5 (GAS5) has been shown to be a regulator for many cancers, including non-small cell lung cancer (NSCLC). Therefore, its role and mechanism in the process of NSCLC deserve to be further revealed. The expression levels of GAS5, fat mass and obesity-associated protein (FTO) and bromodomain-containing protein 4 (BRD4) were detected by quantitative real-time PCR. Western blot analysis was used to examine the protein expression of FTO, BRD4, up-frameshift protein 1 (UPF1) and autophagy-related markers. Methylated RNA immunoprecipitation was used to assess the m6A level of GAS5 regulated by FTO. Cell proliferation and apoptosis were determined using MTT assay, EdU assay and flow cytometry. Autophagy ability was assessed by immunofluorescence staining and transmission electron microscope. Xenograft tumor model was constructed to explore the effects of FTO and GAS5 on NSCLC tumor growth in vivo. The interaction between UPF1 and GAS5 or BRD4 was confirmed by pull-down assay, RIP assay, dual-luciferase reporter assay, and chromatin immunoprecipitation. Fluorescent in situ hybridization was used to analyze the co-localization of GAS5 and UPF1. Actinomycin D treatment was employed to evaluate BRD4 mRNA stability. GAS5 was downregulated in NSCLC tissues and was associated with poor prognosis in NSCLC patients. FTO was highly expressed in NSCLC, and it inhibited GAS5 expression by reducing GAS5 m6A methylation level. GAS5 suppressed by FTO could promote the autophagic death of NSCLC cells in vitro and inhibit NSCLC tumor growth in vivo. In addition, GAS5 was able to interact with UPF1 to reduce the mRNA stability of BRD4. Knockdown of BRD4 reversed the inhibition of GAS5 or UPF1 silencing on the autophagic cell death of NSCLC. The findings of the study showed that lncRNA GAS5 mediated by FTO could contribute to the autophagic cell death of NSCLC by interacting with UPF1 to reduce BRD4 mRNA stability, suggesting that GAS5 might be a vital therapy target for NSCLC progression.

Hypoxia-Induced Neuronal Activity in Glioma Patients Polarizes Microglia by Potentiating RNA m6A Demethylation.

PURPOSE: Neuronal activity in the brain has been reported to promote the malignant progression of glioma cells via nonsynaptic paracrine and electrical synaptic integration mechanisms. However, the interaction between neuronal activity and the immune microenvironment in glioblastoma (GBM) remains largely unclear. EXPERIMENTAL DESIGN: By applying chemogenetic techniques, we enhanced and inhibited neuronal activity in vitro and in a mouse model to study how neuronal activity regulates microglial polarization and affects GBM progression. RESULTS: We demonstrate that hypoxia drove glioma stem cells (GSC) to produce higher levels of glutamate, which activated local neurons. Neuronal activity promoted GBM progression by facilitating microglial M2 polarization through enriching miR-200c-3p in neuron-derived exosomes, which decreased the expression of the m6A writer zinc finger CCCH-type containing 13 (ZC3H13) in microglia, impairing methylation of dual specificity phosphatase 9 (DUSP9) mRNA. Downregulation of DUSP9 promoted ERK pathway activation, which subsequently induced microglial M2 polarization. In the mouse model, cortical neuronal activation promoted microglial M2 polarization whereas cortical neuronal inhibition decreased microglial M2 polarization in GBM xenografts. miR-200c-3p knockdown in cortical neurons impaired microglial M2 polarization and GBM xenograft growth, even when cortical neurons were activated. Treatment with the anti-seizure medication levetiracetam impaired neuronal activation and subsequently reduced neuron-mediated microglial M2 polarization. CONCLUSIONS: These findings indicated that hypoxic GSC-induced neuron activation promotes GBM progression by polarizing microglia via the exosomal miR-200c-3p/ZC3H13/DUSP9/p-ERK pathway. Levetiracetam, an antiepileptic drug, blocks the abnormal activation of neurons in GBM and impairs activity-dependent GBM progression. See related commentary by Cui et al., p. 1073.

Exosome-shuttled FTO from BM-MSCs contributes to cancer malignancy and chemoresistance in acute myeloid leukemia by inducing m6A-demethylation: A nano-based investigation.

Although bone marrow mesenchymal stem cells (BM-MSCs)-derived exosomes have been reported to be closely associated with acute myeloid leukemia (AML) progression and chemo-resistance, but its detailed functions and molecular mechanisms have not been fully delineated. Besides, serum RNA m6A demethylase fat mass and obesity-associated protein (FTO)-containing exosomes are deemed as important indicators for cancer progression, and this study aimed to investigate the role of BM-MSCs-derived FTO-exosomes in regulating the malignant phenotypes of AML cells. Here, we verified that BM-MSCs-derived exosomes delivered FTO to promote cancer aggressiveness, stem cell properties and Cytosine arabinoside (Ara-C)-chemoresistance in AML cells, and the underlying mechanisms were also uncovered. Our data suggested that BM-MSCs-derived FTO-exo demethylated m6A modifications in the m6A-modified LncRNA GLCC1 to facilitate its combination with the RNA-binding protein Hu antigen R (HuR), which further increased the stability and expression levels of LncRNA GLCC1. In addition, LncRNA GLCC1 was verified as an oncogene to facilitate cell proliferation and enhanced Ara-C-chemoresistance in AML cells. Further experiments confirmed that demethylated LncRNA GLCC1 served as scaffold to facilitate the formation of the IGF2 mRNA binding protein 1 (IGF2BP1)-c-Myc complex, which led to the activation of the downstream tumor-promoting c-Myc-associated signal pathways. Moreover, our rescuing experiments validated that the promoting effects of BM-MSCs-derived FTO-exo on cancer aggressiveness and drug resistance in AML cells were abrogated by silencing LncRNA GLCC1 and c-Myc. Thus, the present firstly investigated the functions and underlying mechanisms by which BM-MSCs-derived FTO-exo enhanced cancer aggressiveness and chemo-resistance in AML by modulating the LncRNA GLCC1-IGF2BP1-c-Myc signal pathway, and our work provided novel biomarkers for the diagnosis, treatment and therapy of AML in clinic.

Histone lactylation-boosted ALKBH3 potentiates tumor progression and diminished promyelocytic leukemia protein nuclear condensates by m1A demethylation of SP100A.

Albeit N1-Methyladenosine (m1A) RNA modification represents an important regulator of RNA metabolism, the role of m1A modification in carcinogenesis remains enigmatic. Herein, we found that histone lactylation enhances ALKBH3 expression and simultaneously attenuates the formation of tumor-suppressive promyelocytic leukemia protein (PML) condensates by removing the m1A methylation of SP100A, promoting the malignant transformation of cancers. First, ALKBH3 is specifically upregulated in high-risk ocular melanoma due to excessive histone lactylation levels, referring to m1A hypomethylation status. Moreover, the multiomics analysis subsequently identified that SP100A, a core component for PML bodies, serves as a downstream candidate target for ALKBH3. Therapeutically, the silencing of ALKBH3 exhibits efficient therapeutic efficacy in melanoma both in vitro and in vivo, which could be reversed by the depletion of SP100A. Mechanistically, we found that YTHDF1 is responsible for recognition of the m1A methylated SP100A transcript, which increases its RNA stability and translational efficacy. Conclusively, we initially demonstrated that m1A modification is necessary for tumor suppressor gene expression, expanding the current understandings of dynamic m1A function during tumor progression. In addition, our results indicate that lactylation-driven ALKBH3 is essential for the formation of PML nuclear condensates, which bridges our knowledge of m1A modification, metabolic reprogramming, and phase-separation events.

dCas9-guided demethylation of the AKT1 promoter improves milk protein synthesis in a bovine mastitis mammary gland epithelial model induced by using Staphylococcus aureus.

Mastitis is among the main factors affecting milk quality and yield. Although DNA methylation is associated with mastitis, its role in mastitis remains unclear. In this study, a bovine mastitis mammary epithelial cells (BMMECs) model was established via Staphylococcus aureus infection of bovine mammary gland epithelial cells (BMECs). Bisulfite sequencing PCR was used to determine the methylation status of the AKT1 promoter in BMMECs. We found that the degree of the AKT1 promoter methylation in BMMECs was significantly greater than that in BMECs, and the expression levels of genes related to milk protein synthesis were significantly decreased. We used the pdCas9-C-Tet1-SgRNA 2.0 system to regulate the methylation status of the AKT1 promoter. High-efficiency sgRNAs were screened and dCas9-guided AKT1 promoter demethylation vectors were constructed. Following transfection with the vectors, the degree of methylation of the AKT1 promoter was significantly reduced in BMMECs, while AKT1 protein levels increased. When the methylation level of the AKT1 promoter decreased, the synthesis of milk proteins and the expression levels of genes related to milk protein synthesis increased significantly. The viability of the BMMECs was enhanced. Taken together, these results indicate that demethylation guided by the pdCas9-C-Tet1-SgRNA 2.0 system on the AKT1 promoter can reactivate the expression of AKT1 and AKT1/mTOR signaling pathway-related proteins by reducing the AKT1 promoter methylation level and promoting the recovery milk protein expression in BMMECs, thereby alleviating the symptoms of mastitis.

Demethylation-activated light-up dual-color RNA aptamersensor for label-free detection of multiple demethylases in lung tissues.

Methylation is one of the most prevalent epigenetic modifications in natural organisms, and the processes of methylation and demethylation are closely associated with cell growth, differentiation, gene transcription and expression. Abnormal methylation may lead to various human diseases including cancers. Simultaneous analysis of multiple DNA demethylases remains a huge challenge due to the requirement of diverse substrate probes and scarcity of proper signal transduction strategies. Herein, we propose a sensitive and label-free method for simultaneous monitoring of multiple DNA demethylases on the basis of demethylation-activated light-up dual-color RNA aptamers. The presence of targets AlkB homologue-3 (ALKBH3) and fat mass and obesity-associated enzyme (FTO) erases the methyl group in DNA substrate probes, activating the ligation-mediate bidirectional transcription amplification reaction to produce enormous Spinach and Mango aptamers. The resulting RNA aptamers (i.e., Spinach and Mango aptamers) can bind with their cognate nonfluorescent fluorogens (DFHBI and TO1-biotin) to significantly improve the fluorescence signals. This aptamersensor shows high specificity and sensitivity with a limit of detection (LOD) of 8.50 × 10-14 M for ALKBH3 and 6.80 × 10-14 M for FTO, and it can apply to screen DNA demethylase inhibitors, evaluate DNA demethylase kinetic parameters, and simultaneously measure multiple endogenous DNA demethylases in a single cell. Importantly, this aptamersensor can accurately discriminate the expressions of ALKBH3 and FTO between healthy tissues and non-small cell lung cancer (NSCLC) patient tissues, offering a powerful platform for clinical diagnosis and drug discovery.

The Molecular Basis of Human ALKBH3 Mediated RNA N1 -methyladenosine (m1 A) Demethylation.

N1 -methyladenosine (m1 A) is a prevalent post-transcriptional RNA modification, and the distribution and dynamics of the modification play key epitranscriptomic roles in cell development. At present, the human AlkB Fe(II)/α-ketoglutarate-dependent dioxygenase family member ALKBH3 is the only known mRNA m1 A demethylase, but its catalytic mechanism remains unclear. Here, we present the structures of ALKBH3-oligo crosslinked complexes obtained with the assistance of a synthetic antibody crystallization chaperone. Structural and biochemical results showed that ALKBH3 utilized two ß-hairpins (ß4-loop-ß5 and ß'-loop-ß'') and the α2 helix to facilitate single-stranded substrate binding. Moreover, a bubble-like region around Asp194 and a key residue inside the active pocket (Thr133) enabled specific recognition and demethylation of m1 A- and 3-methylcytidine (m3 C)-modified substrates. Mutation of Thr133 to the corresponding residue in the AlkB Fe(II)/α-ketoglutarate-dependent dioxygenase family members FTO or ALKBH5 converted ALKBH3 substrate selectivity from m1 A to N6 -methyladenosine (m6 A), as did Asp194 deletion. Our findings provide a molecular basis for understanding the mechanisms of substrate recognition and m1 A demethylation by ALKBH3. This study is expected to aid structure-guided design of chemical probes for further functional studies and therapeutic applications.

AlkB-Facilitated Demethylation Enables Quantitative and Site-Specific Detection of Dual Methylation of Adenosine in RNA.

RNA molecules undergo various chemical modifications that play critical roles in a wide range of biological processes. N6,N6-Dimethyladenosine (m6,6A) is a conserved RNA modification and is essential for the processing of rRNA. To gain a deeper understanding of the functions of m6,6A, site-specific and accurate quantification of this modification in RNA is indispensable. In this study, we developed an AlkB-facilitated demethylation (AD-m6,6A) method for the site-specific detection and quantification of m6,6A in RNA. The N6,N6-dimethyl groups in m6,6A can cause reverse transcription to stall at the m6,6A site, resulting in truncated cDNA. However, we found that Escherichia coli AlkB demethylase can effectively demethylate m6,6A in RNA, generating full-length cDNA from AlkB-treated RNA. By quantifying the amount of full-length cDNA produced using quantitative real-time PCR, we were able to achieve site-specific detection and quantification of m6,6A in RNA. Using the AD-m6,6A method, we successfully detected and quantified m6,6A at position 1851 of 18S rRNA and position 937 of mitochondrial 12S rRNA in human cells. Additionally, we found that the level of m6,6A at position 1007 of mitochondrial 12S rRNA was significantly reduced in lung tissues from sleep-deprived mice compared with control mice. Overall, the AD-m6,6A method provides a valuable tool for easy, accurate, quantitative, and site-specific detection of m6,6A in RNA, which can aid in uncovering the functions of m6,6A in human diseases.