AlkB Homolog Research Articles

ALKBH1-mediated m1 A demethylation of METTL3 mRNA promotes the metastasis of colorectal cancer by downregulating SMAD7 expression.

Colorectal cancer (CRC) is one of the most common malignancies, and the main cause of death from CRC is tumor metastasis. m1 A RNA modification plays critical role in many biological processes. However, the role of m1 A modification in CRC remains unclear. Here, we find that the m1 A demethylase alkB homolog 1, histone H2A dioxygenase (ALKBH1) is overexpressed in CRC and is associated with metastasis and poor prognosis. Upregulation of ALKBH1 expression promotes CRC metastasis in vitro and in vivo. Mechanistically, knockdown of ALKBH1 results in a decrease in methyltransferase 3, N6-adenosine-methyltransferase complex catalytic subunit (METTL3) expression, probably due to m1 A modification of METTL3 mRNA, followed by m6 A demethylation of SMAD family member 7 (SMAD7) mRNA. In addition, downregulation of SMAD7 establishes an aggressive phenotype. More importantly, the cell migration and invasion defects caused by ALKBH1 depletion or METTL3 depletion are significantly reversed by SMAD7 silencing. Considering these results collectively, we propose that ALKBH1 promotes CRC metastasis by destabilizing SMAD7 through METTL3.

Genome-Wide Identification and Characterization of the AlkB Gene Family in Sweet Orange (Citrus sinensis).

Sweet orange (Citrus sinensis) is a sub-tropical fruit crop with important economic value that is popular worldwide; however, various pathogens significantly affect citrus cultivation and distribution. AlkB homolog (ALKBH) proteins play crucial roles in RNA metabolism and translation in plants; however, no systematic investigations have been performed on ALKBH in sweet oranges. In this study, ten ALKBH gene family members were identified in Citrus sinensis genome. Standardized analyses, including physical properties, phylogenetic analysis, gene structure, motif composition, cis-acting element prediction, chromosome distribution, and synteny analysis, were conducted. The phylogenetic analysis suggested that the ten proteins were clustered into three groups, each of which had similar motifs and gene structures. Gene expression profiling revealed that almost all CsALKBH proteins were highly expressed in callus, and ALKBH9/10-like group members responded positively to biotic stress. Overall, this study is the first to report a genome-wide assessment of the ALKBH family in sweet oranges and provides valuable insights for candidate gene selection and elucidating the molecular mechanism of sweet orange response to pathogenic infections.

Parent tRNA Modification Status Determines the Induction of Functional tRNA-Derived RNA by Respiratory Syncytial Virus Infection.

tRNA-derived RNA fragments (tRFs) are a recently discovered family of small noncoding RNAs (sncRNAs). We previously reported that respiratory syncytial virus (RSV) infection induces functional tRFs, which are derived from a limited subset of parent tRNAs, in airway epithelial cells. Such induction is also observed in nasopharyngeal wash samples from RSV patients and correlates to RSV genome copies, suggesting a clinical significance of tRFs in RSV infection. This work also investigates whether the modification of parent tRNAs is changed by RSV to induce tRFs, using one of the most inducible tRFs as a model. We discovered that RSV infection changed the methylation modification of adenine at position 57 in tRNA glutamic acid, with a codon of CTC (tRNA-GluCTC), and the change is essential for its cleavage. AlkB homolog 1, a previously reported tRNA demethylase, appears to remove methyladenine from tRNA-GluCTC, prompting the subsequent production of tRFs from the 5'-end of tRNA-GluCTC, a regulator of RSV replication. This study demonstrates for the first time the importance of post-transcriptional modification of tRNAs in tRF biogenesis following RSV infection, providing critical insights for antiviral strategy development.

ALKBH5 attenuates mitochondrial fission and ameliorates liver fibrosis by reducing Drp1 methylation

Mitochondrial metabolism plays a pivotal role in various cellular processes and fibrosis. However, the mechanism underlying mitochondrial metabolic function and liver fibrosis remains poorly understood. In this study, we determined whether mitochondrial metabolism mediates liver fibrosis using cells, animal models, and clinical samples to elucidate the potential effects and underlying mechanism of mitochondrial metabolism in liver fibrosis. We report that AlkB Homolog 5 (ALKBH5) decreases mitochondrial membrane potential (MMP) and oxygen consumption rate (OCR), suppresses mitochondrial fission and hepatic stellate cell (HSC) proliferation and migration and ameliorates liver fibrosis. Enhancement of mitochondrial fission, an essential event during HSC proliferation and migration, is dependent on decreased ALKBH5 expression. Furthermore, we reveal that low ALKBH5 expression is associated with elevated N6-methyladenosine (m6A) mRNA levels. Mechanistically, ALKBH5 mediates m6A demethylation in the 3’UTR of Drp1 mRNA and induces its translation in a YTH domain family proteins 1 (YTHDF1)-independent manner. Subsequently, in transforming growth factor-β1 (TGF-β1) induced HSC, Dynamin-related protein 1 (Drp1) mediates mitochondrial fission and increases cell proliferation and migration. Decreased Drp1 expression inhibits mitochondrial fission and suppresses HSC proliferation and migration. Notably, human fibrotic liver and heart tissue exhibited enhanced mitochondrial fission; increased YTHDF1, Drp1, alpha-smooth muscle actin (α-SMA) and collagen I expression; decreased ALKBH5 expression and increased liver fibrosis. Our results highlight a novel mechanism by which ALKBH5 suppresses mitochondrial fission and HSC proliferation and migration by reducing Drp1 methylation in an m6A-YTHDF1-dependent manner, which may indicate a demethylation-based approach for liver fibrosis diagnosis and therapy.

SMOC2 promotes aggressive behavior of fibroblast-like synoviocytes in rheumatoid arthritis through transcriptional and post-transcriptional regulating MYO1C

Fibroblast-like synoviocytes (FLSs), play a key role in perpetuating synovial inflammation and bone erosion in rheumatoid arthritis (RA), however, the underlying mechanism(s) of RA FLSs activation and aggression remain unclear. Identifying endogenous proteins that selectively target FLSs is urgently needed. Here, we systematically identified that secreted modular calcium-binding protein 2 (SMOC2), was significantly increased in RA FLSs and synovial tissues. SMOC2 knockdown specifically regulated cytoskeleton remodeling and decreased the migration and invasion of RA FLSs. Mechanistically, cytoskeleton-related genes were significantly downregulated in RA FLSs with reduced SMOC2 expression, especially the motor protein myosin1c (MYO1C). SMOC2 controlled MYO1C expression by SRY-related high-mobility group box 4 (SOX4) and AlkB homolog 5 (ALKHB5) mediated-m6A modification through transcriptional and post-transcriptional regulation. Furthermore, intra-articular Ad-shRNA-SMOC2 treatment attenuated synovial inflammation as well as bone and cartilage erosion in rats with collagen-induced arthritis (CIA). Our findings suggest that increased SMOC2 expression in FLSs may contribute to synovial aggression and joint destruction in RA. SMOC2 may serve as a potential target against RA.SMOC2-mediated regulation of the synovial migration and invasion in RA FLSs. In RA FLSs, SMOC2 is significantly increased, leading to the increased level of MYO1C via SOX4-mediated transcriptional regulation and ALKBH5-mediated m6A modification, thereby causing cytoskeleton remodeling and promoting RA FLSs migration and invasion. The Figure was drawn by Figdraw.

Photoperiod alters testicular methyltransferase complex mRNA expression in Siberian hamsters

Exposure to long photoperiods stimulates, whereas exposure to short photoperiods transiently inhibit testicular function in Siberian hamsters via well-described neuroendocrine mechanisms. However, less is known about the intra-testicular regulation of these photoperiod-mediated changes. N6-methyladenosine (m6A) is one of the most common mRNA modifications in eukaryotes, with alterations in m6A mRNA methylation affecting testis function and fertility. We hypothesized that genes controlling m6A methylation such as methyltransferase-like-3 (Mettl3) and −14 (Mettl14) and Wilms’ tumor-1 associated protein (Wtap), part of an mRNA methylating methyl-transferase complex, or the fat-mass-and-obesity-associated (Fto) and the α-ketoglutarate-dependent dioxygenase alkB homolog-5 (Alkbh5) genes responsible for m6A demethylation, may be differentially regulated by photoperiod in the testis. Male hamsters were exposed to long (LD, control) photoperiod for 14-weeks, short (SD) photoperiod for 2, 5, 8, 11 and 14-weeks to induce regression, or SD for 14-weeks followed by transfer to LD for 1, 2, 4 or 8-weeks to induce recrudescence (post-transfer, PT). SD exposure significantly reduced body, testis, and epididymal masses compared to all other groups. Spermatogenic index, seminiferous tubule diameters and testosterone concentrations significantly decreased in SD as compared to LD, returning to levels no different than LD in post-transfer groups. SD exposure significantly decreased Wtap, Fto, Alkbh5, but increased Mettl14 mRNA expression as compared to LD, with values in PT groups restored to LD levels. Mettl3 mRNA expression did not change. These results suggest that testicular recovery induced by stimulatory photoperiod is relatively rapid, and that the methyltransferase complex may play a role during photostimulated testicular recrudescence.

Bone Marrow Mesenchymal Stem Cell-Derived Exosomes Inhibit Triple-Negative Breast Cancer Cell Stemness and Metastasis via an ALKBH5-Dependent Mechanism.

Abnormal N6-methyladenosine (m6A) modification caused by m6A regulators is a common characteristic in various tumors. However, little is known about the role of m6A regulator AlkB homolog 5 (ALKBH5) in triple-negative breast cancer (TNBC). In this study, we analyzed the influence of ALKBH5 on the stemness of TNBC and the molecular mechanism using bioinformatics analysis and in vivo animal experiments. RNA expression data and single-cell RNA sequencing (scRNA-seq) data were downloaded from the TCGA and GEO databases. Following intersection analysis, key genes involved in the TNBC cell stemness were determined, which was followed by functional enrichment analysis, PPI and survival analysis. Exosomes were extracted from bone marrow mesenchymal stem cells (BMSC-Exos) where ALKBH5 inhibition assay was conducted to verify their function in the biological characteristics of TNBC cells. Bioinformatics analysis revealed 45 key genes of ALKBH5 regulating TNBC cell stemness. In addition, UBE2C was predicted as a key downstream gene and p53 was predicted as a downstream signaling of ALKBH5. In vivo data confirmed that ALKBH5 upregulated UBE2C expression by regulating the m6A modification of UBE2C and reduced p53 expression, thus promoting the stemness, growth and metastasis of TNBC cells. BMSC-Exos suppressed the tumor stemness, growth and metastasis of TNBC cells and ALKBH5 shRNA-loaded BMSC-Exos showed a more significant suppressive role. Taken together, our findings indicated that ALKBH5 shRNA-loaded BMSC-Exos reduced TNBC cell stemness, growth and metastasis and define a promising strategy to treat TNBC.

Shikonin targets to m6A-modified oxidative damage pathway to alleviate benzene-induced testicular injury.

Benzene exposure causes reproductive toxicity through oxidative damage. However, the specific mechanisms of benzene-induced testicular damage and the potential therapeutic drugs remain poorly understood. In the present study, C57BL/6J mice have been exposed to 0 and 150mg/kg benzene for four weeks. Then, we found that benzene exposure induced testicular damage in mice, mainly manifested by decreased testicular coefficients, abnormal semen parameters and HE-stained intraepithelial vacuolation. On the mechanism, benzene exposure activated Kelch Like ECH Associated Protein 1/Nuclear factor-erythroid 2 related factor 2 (Keap1/Nrf2) and NF-κB signaling pathway, then promoted apoptosis and inflammatory responses in testes. In vitro, massive reactive oxygen species (ROS) production and a large number of apoptotic cells were observed after 1,4-BQ treatment of GC-2cells. Furthermore, benzene altered the expression of three important RNA methylation modulator genes, methyltransferase-like 3 (Mettl3), AlkB homolog 5 (Alkbh5) and YTH domain containing 2 (Ythdc2). Moreover, both m6A modification and mRNA levels of NF-κB increased with benzene exposure. Inspiringly, shikonin alleviated benzene-induced male reproductive damage by targeting m6A-modified NF-κB in mice testes. Our study provides new insights into molecular mechanisms of RNA m6A modification in benzene-induced reproductive injury and directions to find potential drugs for the treatment of male infertility.

The Role of Hottip Lncrna m6a Modification in AML Genome Organization and Leukemogenesis

Acute myeloid leukemia (AML) is one of the most common and fatal forms of hematopoietic malignancies with the dysregulation of HOX genes. MLLr+ AML is characterized by aberrant expression of HOX genes and the HOX cofactor MEIS1. HOTTIP, a HOXA-associated long non-coding RNA (lncRNA), is significantly up-regulated in MLLr+ AML. Aberrant overexpression of Hottip dramatically perturbs hematopoietic stem cell (HSC) self-renewal leading to mouse AML-like disease through maintaining a leukemic profiling. Recent studies show that N6-methyladenosine (m6A), as the most prevalent internal modification in eukaryotic coding and non-coding RNAs, plays important roles in normal and malignant hematopoiesis. There are several potential m6A modified sites in HOTTIP exon regions and 3'UTR region by re-analyzing m6A-methylated RNA immunoprecipitation sequencing (m6A-meRIP-seq) data in leukemia. However, it is still unclear whether HOTTIP m6A modification plays a critical role in AML genome organization and AML leukemogenesis. Our HOTTIP ChIRP-MASS Spectrometry (ChIRP-MS) data shows that HOTTIP directly interacts with m6A methyltransferase METTL3/METTL14 and CTCF/cohesin chromatin organization proteins in MOLM13 AML cells. To investigate whether HOTTIP lncRNA is methylated by METTL3, we perform m6A-eCLIP-seq and METTL3-eCLIP-seq in MOLM13 cells. Our data indicates that there are several m6A modified sites co-bound with METTL3 in HOTTIP exon and 3'UTR regions. Our m6A-eCLIP-qPCR data further confirms that depletion of METTL3 significantly decreases HOTTIP m6A modification levels at HOTTIP RNA m6A core motif GGACU regions (g1, g2 and g3) by comparing shScramble and shMETTL3 cells. It suggests that HOTTIP lncRNA contains specific m6A modified sites methylated by METTL3. Next, to further examine whether and which HOTTIP m6A sites is required for the interaction of HOTTIP RNA with CTCF protein, we have performed in vitro RNA methylation and RNA binding protein assay by synthesizing biotinylated HOTTIP RNA fragments in g1, g2 and g3 regions. Subsequently, these biotin-HOTTIP-g1/g2/g3 RNAs are methylated with METTL3/METTL14 recombinant proteins and then incubated with GST control or GST-CTCF fusion in vitro. Our data shows that m6A modification of HOTTIP-g2 or HOTTIP-g3 RNA fragment enhances its interaction with GST-CTCF protein, suggesting that HOTTIP m6A modification plays a critical role in coordinating with CTCF-mediated genome organization. To investigate the function of HOTTIP m6A modification in AML leukemogenesis, we have generated CRISPR-dCas13-ALKBH5 (dALK) system to specifically demethylate HOTTIP m6A sites (g1, g2 and g3) by co-transfecting gRNA vector and the catalytically inactive Cas13 (dCas13) fused with m6A demethylase AlkB homolog 5 (ALKBH5) vector. Targeting dALK to specific m6A site in HOTTIP lncRNA results in site-specific loss of m6A modification. Although loss of HOTTIP m6A modification never affects the stability and transcription of HOTTIP in dALK clones, loss of HOTTIP m6A modification significantly affects the transcription of leukemic signature genes HOXA9/13 and WNT target (β-catenin and MYC etc) in dALK-g2 or dALK-g3 cells. Additionally, our HOTTIP ChIRP-qPCR shows that loss of HOTTIP m6A modification affects HOTTIP binding at WNT target loci (β-catenin and MYC loci) in dALK-g2 or dALK-g3 cells. Critically, our data find that loss of HOTTIP m6A modification significantly impairs cell proliferation and cell cycle in dALK-g2 or dALK-g3 cells compared with ctrl cells. To further elucidate whether HOTTIP m6A modification is required for AML leukemogenesis in vivo, we xenograft ctrl and dALK clones into NOD-scid IL2Rgammanull (NSG) mice. Our data indicates that mice transplanted with ctrl or dALK-g1 cells dies around 40 days after transplantation, while those receiving dALK-g2 or dALK-g3 cells survives significantly longer up to 55 days. Critically, we also find that ctrl recipient mice have significantly higher human CD45+ cell chimerism in the BM than mice receiving dALK-g2 or dALK-g3 AML cells. Thus, loss of the specific HOTTIP m6A modification site reduces the AML leukemic burden in vivo. In summary, this research will provide a prospective insight for the role of HOTTIP-m6A-CTCT axis in AML genome organization and leukemogenesis.

ALKBH5-mediated m6A modification of lincRNA LINC02551 enhances the stability of DDX24 to promote hepatocellular carcinoma growth and metastasis

As the most important RNA epigenetic regulation in eukaryotic cells, N6-metheyladenosine (m6A) modification has been demonstrated to play significant roles in cancer progression. However, this modification in long intergenic non-coding RNAs (lincRNAs) and the corresponding functions remain elusive. Here, we showed a lincRNA LINC02551 was downregulated by AlkB Homolog 5 (ALKBH5) overexpression in a m6A-dependent manner in hepatocellular carcinoma (HCC). Functionally, LINC02551 was required for the growth and metastasis of HCC. Mechanistically, LINC02551, a bona fide m6A target of ALKBH5, acted as a molecular adaptor that blocked the combination between DDX24 and a E3 ligase TRIM27 to decrease the ubiquitination and subsequent degradation of DDX24, ultimately facilitating HCC growth and metastasis. Thus, ALKBH5-mediated LINC02551 m6A methylation was required for HCC growth and metastasis.

MiR-654-3p, reduced by the excessive ALKBH5, Alleviated the Inflammation in OA by targeting TNFRSF9, the trigger of the NF-κB pathway

MicroRNA (miRNA) is one of the most potent therapeutic targets for osteoarthritis (OA). We identified that miR-654-3p protected the phenotype of chondrocytes. We demonstrated that TNF receptor superfamily member 9 (TNFRSF9) was the target of miR-654-3p by binding to its 3′UTR regions, based on a dual-luciferase reporter assay and an RNA binding protein immunoprecipitation (RIP) assay. In addition, further experiments proved that TNFRSF9, as a trigger of the NF-κB pathway, correlated with the inflammation in chondrocytes. MiR-654-3p overexpressed in the knee of mice alleviated the OA in vivo. Moreover, we examined the m6A enzyme level in OA, proving that the abnormal expression of α-ketoglutarate-dependent dioxygenase alkB homolog 5 (ALKBH5) contributed to the miR-654-3p decrease. Our research illustrated the significant role of miR-654-3p in OA, including its maturation and the mechanism in protecting the phenotype of chondrocytes, which could be a new treatment target for OA.

ALKBH9C, a potential RNA m6 A demethylase, regulates the response of Arabidopsis to abiotic stresses and abscisic acid.

Although several studies have shown that AlkB homolog (ALKBH) proteins are potential RNA demethylases (referred to as 'erasers'), biological functions of only a few ALKBH proteins have been characterized to date. In this study, we determined the function of ALKBH9C (At4g36090) in seed germination and seedling growth of Arabidopsis thaliana in response to abiotic stress and abscisic acid (ABA). Seed germination of the alkbh9c mutant was delayed in response to salt, drought, coldand ABA. Moreover, seedling growth of the mutant was repressed under salt stress or ABA but enhanced under drought conditions. Notably, the stress-responsive phenotypes were associated with the altered expression of several m6 A-modified transcripts related to salt, droughtor ABA response. Global m6 A levels were increased in the alkbh9c mutant, and ALKBH9C bound to m6 A-modified RNAs and had in vitro m6 A demethylase activity, suggesting its potential role as an m6 A eraser. The m6 A levels in several stress-responsive genes were increased in the alkbh9c mutant, and the stability of m6 A-modified transcripts was altered in the mutant. Collectively, our results suggest that m6 A eraser ALKBH9C is crucial for seed germination and seedling growth of Arabidopsis in response to abiotic stresses or ABA via affecting the stability of stress-responsive transcripts.

Genome-wide identification of the AlkB homologs gene family, PagALKBH9B and PagALKBH10B regulated salt stress response in Populus.

The AlkB homologs (ALKBH) gene family regulates N6-methyladenosine (m6A) RNA methylation and is involved in plant growth and the abiotic stress response. Poplar is an important model plant for studying perennial woody plants. Poplars typically have a long juvenile period of 7–10 years, requiring long periods of time for studies of flowering or mature wood properties. Consequently, functional studies of the ALKBH genes in Populus species have been limited. Based on AtALKBHs sequence similarity with Arabidopsis thaliana, 23 PagALKBHs were identified in the genome of the poplar 84K hybrid genotype (P. alba × P. tremula var. glandulosa), and gene structures and conserved domains were confirmed between homologs. The PagALKBH proteins were classified into six groups based on conserved sequence compared with human, Arabidopsis, maize, rice, wheat, tomato, barley, and grape. All homologs of PagALKBHs were tissue-specific; most were highly expressed in leaves. ALKBH9B and ALKBH10B are m6A demethylases and overexpression of their homologs PagALKBH9B and PagALKBH10B reduced m6A RNA methylation in transgenic lines. The number of adventitious roots and the biomass accumulation of transgenic lines decreased compared with WT. Therefore, PagALKBH9B and PagALKBH10B mediate m6A RNA demethylation and play a regulatory role in poplar growth and development. Overexpression of PagALKBH9B and PagALKBH10B can reduce the accumulation of H2O2 and oxidative damage by increasing the activities of SOD, POD, and CAT, and enhancing protection for Chl a/b, thereby increasing the salt tolerance of transgenic lines. However, overexpression lines were more sensitive to drought stress due to reduced proline content. This research revealed comprehensive information about the PagALKBH gene family and their roles in growth and development and responsing to salt stress of poplar.

LncRNA ALKBH3-AS1 enhances ALKBH3 mRNA stability to promote hepatocellular carcinoma cell proliferation and invasion.

Long noncoding RNAs (lncRNAs) are confirmed as the key regulators of hepatocellular carcinoma (HCC) occurrence and progression, but the role of AlkB homologue 3 antisense RNA 1 (ALKBH3‐AS1) in HCC is unclear. We revealed the overexpression of ALKBH3‐AS1 in HCC tissues. The upregulated levels of ALKBH3‐AS1 were observed in HCC cells. ALKBH3‐AS1 was expressed in the nucleus and cytoplasm of HCC cells. The high ALKBH3‐AS1 expression was markedly associated with a decreased survival rate of HCC patients. ALKBH3‐AS1 knockdown repressed and ALKBH3‐AS1 overexpression enhanced HCC cell invasion and proliferation. ALKBH3‐AS1 silencing restricted HCC growth in vivo. A significant positive correlation between ALKBH3‐AS1 and ALKBH3 mRNA levels was confirmed in HCC specimens. ALKBH3‐AS1 silencing reduced ALKBH3 expression by stabilizing its mRNA stability in HCC cells. Notably, the impact of ALKBH3 silencing on HCC cells was similar to that of ALKBH3‐AS1 knockdown. ALKBH3 restoration prominently attenuated the suppressive effects resulting from ALKBH3‐AS1 silencing in HCCLM3 cells. Hypoxia‐inducible factor‐1α (HIF‐1α) transcriptionally activated ALKBH3‐AS1 expression in hypoxic HCC cells. ALKBH3‐AS1 knockdown markedly attenuated cell proliferation and invasion in hypoxic Huh7 cells. Collectively, HIF‐1α‐activated ALKBH3‐AS1 exerted an oncogenic role by enhancing ALKBH3 mRNA stability in HCC cells.

FTO promotes liver inflammation by suppressing m6A mRNA methylation of IL-17RA.

BackgroundPrevious studies have demonstrated that inflammation-related interleukin-17 (IL-17) signaling plays a pivotal role in the pathogenesis of non-alcoholic steatohepatitis (NASH)- and alcoholic liver disease (ALD)-induced hepatocellular carcinoma (HCC). However, rare efforts have been intended at implementing the analysis of N6-methyladenosine (m6A) mRNA methylation to elucidate the underpinning function of the IL-17 receptor A (IL-17RA) during the inflammation-carcinogenesis transformation of HCC.MethodsWe performed methylated RNA immunoprecipitation sequencing (MeRIP-seq) using normal, HCC tumor and paired tumor adjacent tissues from patients to investigate the dynamic changes of m6A mRNA methylation in the process of HCC. Additionally, murine non-alcoholic fatty liver disease (NAFLD) model and murine chronic liver injury model were utilized to investigate the role of IL-17RA regulated by m6A mRNA modulator fat mass and obesity-associated (FTO) in chronic hepatic inflammation.ResultsMeRIP-seq revealed the reduction of m6A mRNA methylation of IL-17RA in tumor adjacent tissues with chronic inflammation, suggesting the potential role of IL-17RA in the inflammation-carcinogenesis transformation of HCC. Besides, we demonstrated that FTO, rather than methyltransferase-like 3 (METTL3), methyltransferase-like 14 (METTL14), and alkB homolog 5 (ALKBH5) functions as a main modulator for the decrease of m6A mRNA methylation of IL-17RA via knockdown and overexpression of FTO in vitro and in vivo.ConclusionsOverall, we elaborated the underlying mechanisms of the increase of IL-17RA resulting in chronic inflammation via the demethylation of FTO in tumor adjacent tissues and demonstrated that targeting the specific m6A modulator FTO may provide an effective treatment for hepatitis patients to prevent the development of HCC.

Tyrosyl-DNA phosphodiesterase 1 excises the 3′-DNA-ALKBH1 cross-link and its application for 3′-DNA-ALKBH1 cross-link characterization by LC-MS/MS

The apurinic/apyrimidinic (abasic, AP) site is one of the most abundant DNA lesions. Previous studies by others demonstrated that human AlkB homologue 1 (ALKBH1) catalyzes the DNA strand incision at an AP site, resulting in suicidal cross-linking of the enzyme to the 3′-DNA end. Prior site-directed mutagenesis experiments had reported that Cys129 of ALKBH1 is the predominant nucleophile that conjugates to the C3′ position of the incised AP site, 3′-phospho-α,β-unsaturated aldehyde (3′-PUA), to form a 3′-PUA-ALKBH1 cross-link. However, direct evidence to support this mechanism was lacking. The 3′-PUA-ALKBH1 cross-link is so far the only adduct that has been found to form via a Michael addition reaction between a protein and 3′-PUA. It is unclear whether and how this type of cross-link is repaired. In this study, we first demonstrated that the 3′-PUA-ALKBH1 cross-link is fairly stable under physiological temperature and pH as only ~10% of the adduct decomposed after a 3-day incubation. Using a gel-based assay with an aldehyde-reacting probe, we demonstrated that the 3′-PUA-ALKBH1 cross-link has a free aldehyde group that is in line with the Michael addition mechanism. Moreover, we found that the 3′-PUA-ALKBH1 cross-link can be excised by human tyrosyl-DNA phosphodiesterase 1 (TDP1) and the removal efficiency is significantly enhanced if the adduct is pre-digested by trypsin. Notably, we employed TDP1 as a molecular tool to homogeneously release the cross-linked peptides from DNA to facilitate liquid chromatography tandem mass spectrometry analysis, and demonstrated that Cys129 and Cys371 of ALKBH1 cross-link to 3′-PUA.

ALKBH5 Expression could Affect the Function of T Cells in Systemic Lupus Erythematosus Patients: A Case-control Study.

N6-methyladenosine (m6A) modification is widespread in eukaryotic mRNA, regulated by m6A demethylase, AlkB homolog 5 (ALKBH5). However, the role of m6A in systemic lupus erythematosus (SLE) is still obscure. We explored ALKBH5 expression in SLE patients and its effects on T cells. 100 SLE patients and 110 healthy controls were recruited to investigate the expression of ALKBH5 in peripheral blood mononuclear cells (PBMCs). An additional 32 SLE patients and 32 health controls were enrolled to explore the expression of ALKBH5 in T cells. Then we explored the function of ALKBH5 in T cells by lentivirus. The expressions of ALKBH5 were downregulated in both PBMCs and T cells in SLE patients (all P<0.05). In PBMCs: ALKBH5 mRNA levels were associated with a complement C4 level in plasma (P<0.05). In T cells: ALKBH5 mRNA levels were downregulated in SLE patients with low complement levels, high antidsDNA, anti-Sm, anti-RNP, and proteinuria compared with those without, respectively (all P<0.05); ALKBH5 mRNA levels were negatively related with SLE disease activity index score, erythrocyte sedimentation rate, and anti-dsDNA levels (all P<0.05), and positively correlated with complement C3 and C4 level (all P<0.05). Functionally, the overexpression of ALKBH5 promoted apoptosis and inhibited the proliferation of T cells (all P<0.05). ALKBH5 expression is downregulated in SLE patients and could affect the apoptosis and proliferation of T cells, but the exact mechanism still needs to be further explored.

Targeting METTL14 and RNA methylation to treat osteosarcoma.

Targeting METTL14 and RNA methylation to treat osteosarcoma.

Lipid metabolism and m6A RNA methylation are altered in lambs supplemented rumen-protected methionine and lysine in a low-protein diet

BackgroundMethionine or lysine has been reported to influence DNA methylation and fat metabolism, but their combined effects in N6-methyl-adenosine (m6A) RNA methylation remain unclarified. The combined effects of rumen-protected methionine and lysine (RML) in a low-protein (LP) diet on lipid metabolism, m6A RNA methylation, and fatty acid (FA) profiles in the liver and muscle of lambs were investigated. Sixty-three male lambs were divided into three treatment groups, three pens per group and seven lambs per pen. The lambs were fed a 14.5% crude protein (CP) diet (adequate protein [NP]), 12.5% CP diet (LP), and a LP diet plus RML (LP + RML) for 60 d.ResultsThe results showed that the addition of RML in a LP diet tended to lower the concentrations of plasma leptin (P = 0.07), triglyceride (P = 0.05), and non-esterified FA (P = 0.08). Feeding a LP diet increased the enzyme activity or mRNA expression of lipogenic enzymes and decreased lipolytic enzymes compared with the NP diet. This effect was reversed by supplementation of RML with a LP diet. The inclusion of RML in a LP diet affected the polyunsaturated fatty acids (PUFA), n-3 PUFA, and n-6 PUFA in the liver but not in the muscle, which might be linked with altered expression of FA desaturase-1 (FADS1) and acetyl-CoA carboxylase (ACC). A LP diet supplemented with RML increased (P < 0.05) total m6A levels in the liver and muscle and were accompanied by decreased expression of fat mass and obesity-associated protein (FTO) and alkB homologue 5 (ALKBH5). The mRNA expressions of methyltransferase-like 3 (METTL3) and methyltransferase-like 14 (METTL14) in the LP + RML diet group were lower than those in the other two groups. Supplementation of RML with a LP diet affected only liver YTH domain family (YTHDF2) proteins (P < 0.05) and muscle YTHDF3 (P = 0.09), which can be explained by limited m6A-binding proteins that were mediated in mRNA fate.ConclusionsOur findings showed that the inclusion of RML in a LP diet could alter fat deposition through modulations of lipogenesis and lipolysis in the liver and muscle. These changes in fat metabolism may be associated with the modification of m6A RNA methylation.Graphical abstractA systematic graph illustrates the mechanism of dietary methionine and lysine influence on lipid metabolism and M6A. The green arrow with triangular heads indicates as activation and brown-wine arrows with flat heads indicates as suppression.

Hsa_Circ_0000670 Regulates Cell Growth, Angiogenesis and Glutamine Metabolism of Gastric Cancer Cells Through Mir-141-3p/Alkbh1 Axis

Background Gastric cancer (GC) is one of the four most deadly tumors in the world, and its occurrence is influenced by genetic and environmental factors. CircRNA-miRNA-mRNA is reported to be associated with variety cancer, including GC. Methods The expression of hsa_circ_0000670, miR-141-3p and AlkB homolog 1 (ALKBH1) were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Cell proliferation and apoptosis were detected by 5-Ethynyl2’-deoxyuridine (EdU) assay and flow cytometry. The protein expression levels were measured by western blot and immunohistochemistry (IHC). Tube forming experiment was performed to assess angiogenesis rate. The levels of glutamine and α-ketoglutaric acid (α-KG) were examined using commercial kits. The target interaction between miR-141-3p and circ_0000670 or ALKBH1 was verified by dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. The effect of circ_0000670 on tumor in vivo was analyzed in mice. Results Circ_0000670 had a stable ring structure and was accelerated in GC tissues and cells. The proliferation of GC was hampered by circ_0000670 knockdown in vivo and in vitro, and the apoptosis rate was increased by circ_0000670 knockdown. Circ_0000670 down-regulation suppressed angiogenesis, the level of glutamine and α-KG in GC cells. The impacts of circ_0000670 knockdown on GC were relieved by miR-141-3p inhibition. The results indicated that circ_0000670 silencing could induce cell proliferation decline in GC, which was ameliorated by miR-141-3p knockdown. The functions of miR-141-3p mimic on cell proliferation, apoptosis, angiogenesis, glutamine and α-KG were reversed by ALKBH1 up-regulation. Conclusion Our findings revealed that circ_0000670 promoted GC progression though miR-141-3p and ALKBH1, suggesting that circ_0000670 might be a target for GC clinical treatment.