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

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. 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). 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). 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.

BackgroundHepatitis B Virus (HBV) contributes to liver carcinogenesis via various epigenetic mechanisms. The newly defined epigenetics, epitranscriptomics regulation, has been reported to involve in multiple cancers including Hepatocellular Carcinoma (HCC). Our previous study found that HBx, HBV encodes X protein, mediated H3K4me3 modification in WDR5-dependent manner to involve in HBV infection and contribute to oncogene expression. AlkB Homolog 5 (ALKBH5), one of epitranscriptomics enzymes, has been identified to be associated with various cancers. However, whether and how ALKBH5 is dysregulated in HBV-related HCC remains unclear yet. This study aims to investigate ALKBH5 function, clinical significance and mechanism in HBV related HCC (HBV-HCC) patients derived from Chinese people.MethodsThe expression pattern of ALKBH5 was evaluated by RT-qPCR, Western blot, data mining and immunohistochemistry in total of 373 HBV-HCC tissues and four HCC cell lines. Cell Counting Kit 8 (CCK8) assay, Transwell and nude mouse model were performed to assess ALKBH5 function by both small interference RNAs and lentiviral particles. The regulation mechanism of ALKBH5 was determined in HBx and WDR5 knockdown cells by CHIP-qPCR. The role of ALKBH5 in HBx mRNA N6-methyladenosine (m6A) modification was further evaluated by MeRIP-qPCR and Actinomycin D inhibitor experiment in HBV-driven cells and HBx overexpression cells.ResultALKBH5 increased in tumor tissues and predicts a poor prognosis of HBV-HCC. Mechanically, the highly expressed ALKBH5 is induced by HBx-mediated H3K4me3 modification of ALKBH5 gene promoter in a WDR5-dependent manner after HBV infection. The increased ALKBH5 protein catalyzes the m6A demethylation of HBx mRNA, thus stabilizing and favoring a higher HBx expression level. Furthermore, there are positive correlations between HBx and ALKBH5 in HBV-HCC tissues, and depletion of ALKBH5 significantly inhibits HBV-driven tumor cells’ growth and migration in vitro and in vivo.ConclusionsHBx-ALKBH5 may form a positive-feedback loop to involve in the HBV-induced liver carcinogenesis, and targeting the loop at ALKBH5 may provide a potential way for HBV-HCC treatment.

ALKBH5 promotes cardiac fibroblasts pyroptosis after myocardial infarction through Notch1/NLRP3 pathway.

Colorectal cancer (CRC) is the major subtype of gastrointestinal malignancy and involves cancer-related genes and signaling pathways to regulate ferroptosis. The present study was conducted to analyze the role of alkB homolog 5 (ALKBH5) in the ferroptosis of CRC cells and provide novel targets for CRC treatment. The transcriptional and protein levels of ALKBH5 and solute carrier family 7 members 11 (SLC7A11) in tissues and cells were determined by qRT-PCR and Western blot assay. HCT116 and SW620 cells were transfected with ALKBH5 overexpression vectors to determine cell viability and levels of reactive oxygen species (ROS), Fe+, glutathione, and glutathione peroxidase 4 using cell counting kit-8, colony formation, fluorescence probe, assay kits, and Western blot assay. The N6-methyladenosine (m6A) level and the enrichment of m6A on SLC7A11 mRNA were measured by m6A quantitative analysis and m6A methylated RNA immunoprecipitation-qPCR, and the mRNA stability was determined after actinomycin D treatment. CRC cells were treated with the combination of SLC7A11 and ALKBH5 overexpression vectors to confirm the mechanism. Nude mice were subcutaneously injected with CRC cells overexpressing ALKBH5. ALKBH5 was downregulated in CRC and ALKBH5 overexpression promoted ROS release and ferroptosis. ALKBH5 erased the m6A modification on SLC7A11 mRNA to reduce the mRNA stability of SLC7A11, further reducing SLC7A11 expression. SLC7A11 overexpression reversed the promotive role of ALKBH5 overexpression in ferroptosis. ALKBH5 upregulation mitigated tumor growth in vivo. ALKBH5 reduced SLC7A11 transcription by erasing m6A modification, thus promoting the ferroptosis of CRC cells.

BackgroundBreast cancer is the most prevalent disease and the fourth cause of cancer death among female globally. The N6-methyladenylate methylation (m6A) demethylase alpha-ketoglutarate-dependent dioxygenase alkB homolog 5 (ALKBH5) decreases modification of RNA, while its role in regulating breast cancer development remains unclear.MethodsALKBH5-silenced breast cancer cell-line MCF-7 was constructed to investigate its functional impact. Cell proliferation, migration and invasion ability were evaluated by CCK8 and transwell assays under ALKBH5 inhibition. Spheroid formation and in vitro extreme limiting dilution analysis (ELDA) were performed to elucidate the effect of ALKBH5 deficiency on stemness of MCF-7 cells. The m6A modification level of CTNNB1 and the interaction of ALKBH5 and CTNNB1 were investigated by Methylated RNA immunoprecipitation (MeRIP) and RIP assay respectively.ResultsSilencing ALKBH5 significantly suppressed MCF-7 cell proliferation, migration, and invasion abilities. Moreover, ALKBH5 depletion also diminished the stemness of breast cancer cells in vitro. Further investigation illustrated that ALKBH5 may regulate Wnt/β-catenin signaling via an m6A-dependant manner. Clinical data analysis demonstrated a strong positive relationship between ALKBH5 and β-catenin expression.ConclusionThis study establishes a link between ALKBH5 and cancer stemness in breast cancer, providing insights into the functional role of demethylase ALKBH5 in breast cancer progression.

Coronary heart disease (CHD) occurs when the arteries supplying blood to the heart become narrowed or blocked owing to plaque buildup, leading to reduced blood flow and potential heart attacks. N6-methyladenosine (m6A) modification is a common form of post-transcriptional RNA modification. ALKB homolog 5 (ALKBH5) is an RNA demethylase that specifically removes the m6A modification from RNA. This study aimed to investigate the role of ALKBH5 in CHD and the underlying mechanism. Both cellular and animal CHD models were established. The expression levels of Alkbh5 and fibrosis-related markers were analyzed by qRT-PCR. Cell viability and cytotoxicity were assessed via cell counting kit-8. Inflammatory cytokines levels were detected by ELISA. Flow cytometry was used to detect pyroptosis. The interaction between ALKBH5/YTH N6-methyladenosine RNA binding protein (YTHDF)1 and SPEN transcriptional repressor (SPEN) was examined through RNA immunoprecipitation and dual-luciferase reporter assays. ALKBH5-mediated demethylation of m6A was decreased in CHD rat heart tissues and oxidized low-density lipoprotein (ox-LDL)-treated H9c2 cells. In addition, Alkbh5 overexpression increased the cell viability and suppressed the inflammation, pyroptosis, and fibrosis in ox-LDL-treated H9c2 cells. In in vivo studies, Alkbh5 overexpression reduced myocardial injury and fibrosis in CHD rats by suppressing inflammation and pyroptosis. Mechanically, Alkbh5 overexpression decreased the stability of Spen mRNA. Additionally, ALKBH5/YTHDF1 m6A axis regulated the expression of Spen. Moreover, Ythdf1 overexpression counteracted ALKBH5-mediated inhibition of inflammation, pyroptosis, and fibrosis in ox-LDL-treated H9c2 cells. ALKBH5 regulated inflammation and pyroptosis of CHD by targeting SPEN in a YTHDF1-mediated manner, which could provide a reference for CHD treatment.

Lung cancer is the leading cause of cancer-related mortality worldwide, accounting for 18.4% of all cancer deaths. This study aims to investigate the underlying mechanism by which long non-coding RNA (lncRNA) ENST00000425005 mediates doxorubicin resistance and epithelial-mesenchymal transition (EMT) in lung cancer cells. The expression levels of ENST00000425005 in non-small cell lung cancer (NSCLC) cells were determined using quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). The protein levels of alkB homolog 5 (ALKBH5) and EMT markers (including Snail1, E-cadherin, N-cadherin, and Vimentin) were assessed using Western Blot analysis. RNA binding protein immunoprecipitation assays were conducted to detect the interaction between ENST00000425005 and ALKBH5. Cell viability was evaluated using cell counting kits assay, and cell invasion was determined by transwell assay. It was found that ENST00000425005 expression was downregulated, while ALKBH5 expression was upregulated in NSCLC cells. Additionally, ALKBH5 bound to ENST00000425005 and downregulated its expression. Overexpression of ALKBH5 reduced m6A modification and RNA levels of ENST00000425005. Moreover, co-overexpression of ENST00000425005 and ALKBH5 rescued loss of NSCLC cell viability, invasion, and doxorubicin resistance caused by overexpression of ENST00000425005. Furthermore, this co-overexpression rescued ENST00000425005-induced changes in expression of E-cadherin, Snail1, N-cadherin, and Vimentin. The reduction of m6A methylation modification on lncRNA ENST00000425005 caused by binding to ALKBH5 promoted doxorubicin resistance and EMT progression in NSCLC cells. In summary, targeting lncRNA ENST00000425005 holds promise as a therapeutic strategy for NSCLC.

Upstream-stimulating factor 1 (USF1) is a ubiquitously expressed transcription factor implicated in multiple cellular processes, including metabolism and proliferation. This study focused on the function of USF1 in glycolysis and the malignant development of prostate adenocarcinoma (PRAD). Bioinformatics predictions suggested that USF1 is poorly expressed in PRAD. The clinical PRAD samples revealed a low level of USF1, which was correlated with an unfavorable prognosis. Artificial upregulation of USF1 significantly repressed glycolytic activity in PRAD cells and reduced cell growth and metastasis in vitro and in vivo. Potential downstream genes of USF1 were probed by integrated bioinformatics analyses. The chromatin immunoprecipitation and luciferase assays indicated that USF1 bound to the α-ketoglutarate-dependent dioxygenase alkB homolog 5 (ALKBH5) promoter for transcription activation. Flightless I (FLII) was identified as the gene showing the highest degree of correlation with ALKBH5. As an m6A demethylase, ALKBH5 enhanced FLII mRNA stability by inducing m6A demethylation in an m6A-YTH N6-methyladenosine RNA-binding protein F2 (YTHDF2)-dependent manner. Either silencing of ALKBH5 or FLII blocked the role of USF1 in PARD cells and restored glycolysis, cell proliferation, and invasion. This study demonstrates that USF1 activates ALKBH5 to stabilize FLII mRNA in an m6A-YTHDF2-dependent manner, thereby repressing glycolysis processes and the progression of PRAD.

Myocardial ischemia‒reperfusion injury (MI/RI) is closely related to pyroptosis. alkB homolog 5 (ALKBH5) is abnormally expressed in the MI/RI models. However, the detailed molecular mechanism of ALKBH5 in MI/RI has not been elucidated. In this study, rats and H9C2 cells served as experimental subjects and received MI/R induction and H/R induction, respectively. The abundance of the targeted molecules was evaluated using RT-qPCR, Western blotting, immunohistochemistry, immunofluorescence, and enzyme-linked immunosorbent assay. The heart functions of the rats were evaluated using echocardiography, and heart injury was evaluated. Cell viability and pyroptosis were determined using cell counting Kit-8 and flow cytometry, respectively. Total m6A modification was measured using a commercial kit, and pri-miR-199a-5p m6A modification was detected by Me-RNA immunoprecipitation (RIP) assay. The interactions among the molecules were validated using RIP and luciferase experiments. ALKBH5 was abnormally highly expressed in H/R-induced H9C2 cells and MI/RI rats. ALKBH5 silencing improved injury and inhibited pyroptosis. ALKBH5 reduced pri-miR-199a-5p m6A methylation to block miR-199a-5p maturation and inhibit its expression. TNF receptor-associated Factor 3 (TRAF3) is a downstream gene of miR-199a-5p. Furthermore, in H/R-induced H9C2 cells, the miR-199a-5p inhibitor-mediated promotion of pyroptosis was reversed by ALKBH5 silencing, and the TRAF3 overexpression-mediated promotion of pyroptosis was offset by miR-199a-5p upregulation. ALKBH5 silencing inhibited pri-miR-199a-5p expression and enhanced pri-miR-199a-5p m6A modification to promote miR-199a-5p maturation and enhance its expression, thereby suppressing pyroptosis to alleviate MI/RI through decreasing TRAF3 expression.

Bladder cancer (BLCA) is a prevalent and life-threatening condition that significantly impacts patients' quality of life while imposing substantial financial costs on healthcare systems. Advancing our knowledge of the mechanisms underlying tumor development is crucial for improving treatment outcomes. Emerging studies emphasize the critical role of the RNA modification 6-methyladenine (m6A) and its associated proteins, methyltransferase-like 3 (METTL3), Vir-like m6A methyltransferase associated (VIRMA) (writers), Alkb homolog 5 (ALKBH5) and fat mass and obesity associated protein (FTO) (erasers), in maintaining m6A homeostasis. Dysregulation of these enzymes leads to aberrant m6A methylation, a hallmark of various cancers, including BLCA. Furthermore, m6A modifications influence cisplatin sensitivity, a key drug in muscle-invasive bladder cancer (MIBC) treatment. With this background, we investigated the combined effects of ALKBH5 and FTO knock-down in bladder tumor cell lines. We first investigated the expression of METTL3, VIRMA, ALKBH5 and FTO in BLCA tissues and human bladder tumor cell lines from urinary cancer cells by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Simultaneous knock-down of the expression of the erasers was then performed to explore their consequences in bladder cells. We then conducted cisplatin and mitomycin (MMC) treatment in knock-down cells to decipher the effect of their reduction. The cell viability was evaluated with cell counting kit-8 (CCK-8) assay after the two treatment regimes. Lower expression of ALKBH5 and FTO was identified in BLCA tissue and bladder tumor cell lines. Notably, this trend was consistent across both low-grade and high-grade tissue samples. Furthermore, lower expression levels of ALKBH5 and FTO were observed in tumor cell lines derived from both men and women compared to the non-tumorigenic SV-HUC1 cell line. In contrast, both tissue and cell line data revealed an increased expression tendency of the m6A writers METTL3 and VIRMA. Additionally, knock-down of the two m6A erasers was found to enhance tolerance to cisplatin and MMC treatment, resulting in increased resistance to cell death. Our findings reveal that ALKBH5 and FTO are down-regulated in BLCA and their knock-down confers resistance to cisplatin and MMC in vitro. This suggests that m6A erasers play a critical role in modulating chemotherapy sensitivity, potentially serving as biomarkers or therapeutic targets for enhancing treatment efficacy in BLCA.

Melanoma is a highly malignant skin cancer. This study aimed to investigate the role of long non-coding RNA MIR205 host gene (lncRNA MIR205HG) in proliferation, invasion, and migration of melanoma cells via jumonji domain containing 2C (JMJD2C) and ALKB homolog 5 (ALKBH5). Real-time quantitative polymerase chain reaction or Western blot assay showed that MIR205HG, JMJD2C, and ALKBH5 were increased in melanoma cell lines. Cell counting kit-8, colony formation, and Transwell assays showed that silencing MIR205HG inhibited proliferation, invasion, and migration of melanoma cells. RNA immunoprecipitation, actinomycin D treatment, and chromatin immunoprecipitation showed that MIR205HG may bind to human antigen R (HuR, ELAVL1) and stabilized JMJD2C expression, and JMJD2C may increase the enrichment of H3K9me3 in the ALKBH5 promotor region to promote ALKBH5 transcription. The tumor xenograft assay based on subcutaneous injection of sh-MIR205HG-treated melanoma cells showed that silencing MIR205HG suppressed tumor growth and reduced Ki67 positive rate by inactivating the JMJD2C/ALKBH5 axis. Generally, MIR205HG facilitated proliferation, invasion, and migration of melanoma cells through HuR-mediated stabilization of JMJD2C and increasing ALKBH5 transcription by erasing H3K9me3.

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

Asthma is a chronic respiratory disease that is characterized by airway inflammation, excessive mucus production, and airway remodeling. Prevention and treatment for asthma is an urgent issue in clinical studies. In recent years, N6-methyladenosine methylation (m6A) has emerged as a promising regulatory approach involved in multiple diseases. ALKBH5 (alkB homolog 5) is a demethylase widely studied in disease pathologies. This work aimed to explore the regulatory mechanisms underlying the ALKBH5-regulated asthma. We established an interleukin-13 (IL-13)-stimulated cell model to mimic the in vitro inflammatory environment of asthma. ALKBH5 knockdown in bronchial epithelial cells was performed using siRNAs, and the knockdown efficacy was analyzed by quantitative PCR (qPCR). Cell viability and proliferation were measured by cell counting kit 8 (CCK-8) and colony formation assay. The ferroptosis was assessed by measuring the total iron, Fe2+, lipid reactive oxygen species (ROS), malondialdehyde (MDA), and superoxide dismutase (SOD) levels. The enrichment of N6-methyladenosine methylation (m6A) modification was detected by the MeRIP assay. Knockdown of ALKBH5 significantly elevated the survival and colony formation ability of bronchial epithelial cells in the IL-13 induction model. The levels of total iron, Fe2+, lipid ROS, and MDA were remarkedly elevated, and the SOD level was reduced in IL-13-induced bronchial epithelial cells, and depletion of ALKBH5 reversed these effects. Knockdown of ALKBH5 elevated the enrichment of m6A modification and expression of glutathione peroxidase 4 (GPX4). Knockdown of GPX4 abolished the pro-proliferation and anti-ferroptosis effects of siALKBH5. Knockdown of ALKBH5 improved the proliferation of bronchial epithelial cells and alleviated cell ferroptosis.

ALKB homolog 5 (ALKBH5)-induced circPUM1 upregulation facilitated the progression of neuroblastoma via miR-423–5p/PA2G4 axis

m6A modification impacts hepatic drug and lipid metabolism properties by regulating carboxylesterase 2