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Background Histone lactylation, a metabolic stress-related histone modification, plays an important role in the regulation of gene expression during M1 macrophage polarization. However, the role of histone lactylation in tumorigenesis remains unclear. Results Here, we show histone lactylation is elevated in tumors and is associated with poor prognosis of ocular melanoma. Target correction of aberrant histone lactylation triggers therapeutic efficacy both in vitro and in vivo. Mechanistically, histone lactylation contributes to tumorigenesis by facilitating YTHDF2 expression. Moreover, YTHDF2 recognizes the m6A modified PER1 and TP53 mRNAs and promotes their degradation, which accelerates tumorigenesis of ocular melanoma. Conclusion We reveal the oncogenic role of histone lactylation, thereby providing novel therapeutic targets for ocular melanoma therapy. We also bridge histone modifications with RNA modifications, which provides novel understanding of epigenetic regulation in tumorigenesis.

Background N6-methyladenosine (m 6 A) is the most abundant modification in mRNA of humans. Emerging evidence has supported the fact that m 6 A is comprehensively involved in various diseases especially cancers. As a crucial reader, YTHDF2 usually mediates the degradation of m 6 A-modified mRNAs in m 6 A-dependent way. However, the function and mechanisms of m 6 A especially YTHDF2 in prostate cancer (PCa) still remain elusive. Methods To investigate the functions and mechanisms of YTHDF2 in PCa, in vitro , in vivo biofunctional assays and epigenetics experiments were performed. Endogenous expression silencing of YTHDF2 and METTL3 was established with lentivirus-based shRNA technique. Colony formation, flow cytometry and trans-well assays were performed for cell function identifications. Subcutaneous xenografts and metastatic mice models were combined with in vivo imaging system to investigate the phenotypes when knocking down YTHDF2 and METTL3. m 6 A RNA immunoprecipitation (MeRIP) sequencing, mRNA sequencing, RIP-RT-qPCR and bioinformatics analysis were mainly used to screen and validate the direct common targets of YTHDF2 and METTL3. In addition, TCGA database was also used to analyze the expression pattern of YTHDF2, METTL3 and the common target LHPP in PCa, and their correlation with clinical prognosis. Results The upregulated YTHDF2 and METTL3 in PCa predicted a worse overall survival rate. Knocking down YTHDF2 or METTL3 markedly inhibited the proliferation and migration of PCa in vivo and in vitro. LHPP and NKX3–1 were identified as the direct targets of both YTHDF2 and METTL3. YTHDF2 directly bound to the m 6 A modification sites of LHPP and NKX3–1 to mediate the mRNA degradation. Knock-down of YTHDF2 or METTL3 significantly induced the expression of LHPP and NKX3–1 at both mRNA and protein level with inhibited phosphorylated AKT. Overexpression of LHPP and NKX3–1 presented the consistent phenotypes and AKT phosphorylation inhibition with knock-down of YTHDF2 or METTL3. Phosphorylated AKT was consequently confirmed as the downstream of METTL3/YTHDF2/LHPP/NKX3–1 to induce tumor proliferation and migration. Conclusion We propose a novel regulatory mechanism in which YTHDF2 mediates the mRNA degradation of the tumor suppressors LHPP and NKX3–1 in m 6 A-dependent way to regulate AKT phosphorylation-induced tumor progression in prostate cancer. We hope our findings may provide new concepts of PCa biology.

Endometriosis (EMs) is a benign gynecological disorder that exhibits several malignant characteristics, including proliferation and angiogenesis. N6-methyladenosine (m6A) modification plays a crucial role in regulating RNA splicing, subcellular localization, translation and RNA-protein interactions, ensuring precise and timely gene expression. Despite ongoing research, the pathogenesis of EMs remains elusive. This study aimed to investigate the potential roles of m6A regulators N6-adenosine-methyltransferase catalytic subunit (METTL3) and YTH domain family, member 2 (YTHDF2) in the development of EMs. We employed a comprehensive approach that combi data from the Gene Expression Omnibus (GEO) database, biological information analysis technologies, and validation using other databases and clinical tissues. This allowed us to uncover aberrantly m6A regulators METTL3 and YTHDF2 and investigate biological functions of EMs. Our investigation identified METTL3 and YTHDF2 as critical m6A regulators exhibiting significant downregulation in ectopic endometrium samples compared to healthy controls. In vitro of biological behaviors studies demonstrated that METTL3 and YTHDF2 inhibited proliferation, migration, invasion and angiogenesis. These findings unveil novel insights into m6A modification of EMs, shedding light on potential biomarkers and paving the way for precision medicine approaches in the treatment for EMs.

The YTH N 6 -methyladenosine RNA binding proteins (YTHDFs) mediate the functional effects of N 6 -methyladenosine (m 6 A) on RNA. Recently, a report proposed that all YTHDFs work redundantly to facilitate RNA decay, raising questions about the exact functions of individual YTHDFs, especially YTHDF1 and YTHDF2. We show that YTHDF1 and YTHDF2 differ in their low-complexity domains (LCDs) and exhibit different behaviors in condensate formation and subsequent physiological functions. Biologically, we also find that the global stabilization of RNA after depletion of all YTHDFs is driven by increased P-body formation and is not strictly m 6 A dependent.

Background Dynamic N 6 -methyladenosine (m 6 A) modification was previously identified as a ubiquitous post-transcriptional regulation that affected mRNA homeostasis. However, the m 6 A-related epitranscriptomic alterations and functions remain elusive in human cancer. Here we aim to identify the profile and outcome of m 6 A-methylation in hepatocellular carcinoma (HCC). Results Using liquid chromatography-tandem mass spectrometry and m 6 A-immunoprecipitation in combination with high-throughput sequencing, we determined the m 6 A-mRNA levels in human HCC. Human HCC exhibited a characteristic gain of m 6 A modification in tandem with an increase of mRNA expression, owing to YTH domain family 2 (YTHDF2) reduction. The latter predicted poor classification and prognosis of HCC patients, and highly correlated with HCC m 6 A landscape. YTHDF2 silenced in human HCC cells or ablated in mouse hepatocytes provoked inflammation, vascular reconstruction and metastatic progression. Mechanistically, YTHDF2 processed the decay of m 6 A-containing interleukin 11 (IL11) and serpin family E member 2 (SERPINE2) mRNAs, which were responsible for the inflammation-mediated malignancy and disruption of vascular normalization. Reciprocally, YTHDF2 transcription succumbed to hypoxia-inducible factor-2α (HIF-2α). Administration of a HIF-2α antagonist (PT2385) restored YTHDF2-programed epigenetic machinery and repressed liver cancer. Conclusion Our results have characterized the m 6 A-mRNA landscape in human HCC and revealed YTHDF2 as a molecular ‘rheostat’ in epitranscriptome and cancer progression.

Background Colorectal cancer (CRC) is one of the leading causes of tumor-related death worldwide, and its main cause of death is distant metastasis. Methyltransferase-like 14(METTL14), a major RNA N6-adenosine methyltransferase, is involved in tumor progression via regulating RNA function. The goal of the study is to uncover the biological function and molecular mechanism of METTL14 in CRC. Methods Quantitative real-time PCR (qRT-PCR), western blot and immunohistochemical (IHC) assays were employed to detect METTL14 and SOX4 in CRC cell lines and tissues. The biological functions of METTL14 were demonstrated using in vitro and in vivo experiments. Chromatin immunoprecipitation (ChIP), Transcrptomic RNA sequencing (RNA-Seq), m6A-RNA immunoprecipitation sequencing (MeRIP-Seq), RNA immunoprecipitation and luciferase reporter assays were used to explore the mechanism of METTL14 action. Results METTL14 expression was significantly downregulated in CRC and decreased METTL14 was associated with poor overall survival (OS). Both the univariate and multivariate Cox regression analysis indicated that METTL14 was an independent prognostic factor in CRC. Moreover, lysine-specific histone demethylase 5C(KDM5C)-mediated demethylation of histone H3 lysine 4 tri-methylation(H3K4me3) in the promoter of METTL14 inhibited METTL14 transcription. Functionally, we verified that METTL14 inhibited CRC cells migration, invasion and metastasis through in vitro and in vivo assays, respectively. Furthermore, we identified SRY-related high-mobility-group box 4(SOX4) as a target of METTL14-mediated m6A modification. Knockdown of METTL14 markedly abolished SOX4 mRNA m6A modification and elevated SOX4 mRNA expression. We also revealed that METTL14-mediated SOX4 mRNA degradation relied on the YTHDF2-dependent pathway. Lastly, we demonstrated that METTL14 might inhibit CRC malignant process partly through SOX4-mediated EMT process and PI3K/Akt signals. Conclusions Decreased METTL14 facilitates tumor metastasis in CRC, suggesting that METTL14 might be a potential prognostic biomarker and effective therapeutic target for CRC. Graphical abstract

N6‐Methyladenosine (m6A) modification, the most prevalent modification of eukaryotic messenger RNA (mRNA), is involved in the progression of various tumours. However, the specific role of m6A in bladder cancer (BCa) is still poorly understood. In this study, we demonstrated the tumour‐promoting function and specific regulatory mechanism of m6A axis, consisting of the core ‘writer’ protein METTL3 and the major reader protein YTHDF2. Depletion of METTL3 impaired cancer proliferation and cancer metastasis in vitro and in vivo. Through transcriptome sequencing, m6A methylated RNA immunoprecipitation (MeRIP) and RIP, we determined that the METTL3/YTHDF2 m6A axis directly degraded the mRNAs of the tumour suppressors SETD7 and KLF4, contributing to the progression of BCa. In addition, overexpression of SETD7 and KLF4 revealed a phenotype consistent with that induced by depletion of the m6A axis. Thus, our findings on the METTL3/YTHDF2/SETD7/KLF4 m6A axis provide the insight into the underlying mechanism of carcinogenesis and highlight potential therapeutic targets for BCa.

Background The tumor suppressor FBW7 is the substrate recognition component of the SCF E3-ubiquitin ligase complex that mediates proteolytic degradation of various oncogenic proteins. However, the role of FBW7 in ovarian cancer progression remains inadequately understood. Methods IP-MASS, co-IP, immunohistochemistry, and western blotting were used to identify the potential substrate of FBW7 in ovarian cancer. The biological effects of FBW7 were investigated using in vitro and in vivo models. LC/MS was used to detect the m 6 A levels in ovarian cancer tissues. MeRIP-Seq and RNA-Seq were used to assess the downstream targets of YTHDF2. Results We unveil that FBW7 is markedly down-regulated in ovarian cancer tissues and its high expression is associated with favorable prognosis and elevated m 6 A modification levels. Consistently, ectopic FBW7 inhibits ovarian cancer cell survival and proliferation in vitro and in vivo, while ablation of FBW7 empowers propagation of ovarian cancer cells. In addition, the m 6 A reader protein, YTHDF2, is identified as a novel substrate for FBW7. FBW7 counteracts the tumor-promoting effect of YTHDF2 by inducing proteasomal degradation of the latter in ovarian cancer. Furthermore, YTHDF2 globally regulates the turnover of m 6 A-modified mRNAs, including the pro-apoptotic gene BMF. Conclusions Our study has demonstrated that FBW7 suppresses tumor growth and progression via antagonizing YTHDF2-mediated BMF mRNA decay in ovarian cancer.

Background N 6 -methyladenosine (m 6 A) modification in mRNAs was recently shown to be dynamically regulated, indicating a pivotal role in multiple developmental processes. Most recently, it was shown that the Mettl3-Mettl14 writer complex of this mark is required for the temporal control of cortical neurogenesis. The m 6 A reader protein Ythdf2 promotes mRNA degradation by recognizing m 6 A and recruiting the mRNA decay machinery. Results We show that the conditional depletion of the m 6 A reader protein Ythdf2 in mice causes lethality at late embryonic developmental stages, with embryos characterized by compromised neural development. We demonstrate that neural stem/progenitor cell (NSPC) self-renewal and spatiotemporal generation of neurons and other cell types are severely impacted by the loss of Ythdf2 in embryonic neocortex. Combining in vivo and in vitro assays, we show that the proliferation and differentiation capabilities of NSPCs decrease significantly in Ythdf2 −/− embryos. The Ythdf2 −/− neurons are unable to produce normally functioning neurites, leading to failure in recovery upon reactive oxygen species stimulation. Consistently, expression of genes enriched in neural development pathways is significantly disturbed. Detailed analysis of the m 6 A-methylomes of Ythdf2 −/− NSPCs identifies that the JAK-STAT cascade inhibitory genes contribute to neuroprotection and neurite outgrowths show increased expression and m 6 A enrichment. In agreement with the function of Ythdf2, delayed degradation of neuron differentiation-related m 6 A-containing mRNAs is seen in Ythdf2 −/− NSPCs. Conclusions We show that the m 6 A reader protein Ythdf2 modulates neural development by promoting m 6 A-dependent degradation of neural development-related mRNA targets.

Background Hypoxia, a typical hallmark of solid tumors, exhibits an essential role in the progression of colorectal cancer (CRC), in which the dysregulation of long non-coding RNAs (lncRNAs) is frequently observed. However, the underlying mechanisms are not clearly defined. Methods The TCGA database was analyzed to identify differential lncRNA expression involved in hypoxia-induced CRC progression. qRT-PCR was conducted to validate the upregulation of lncRNA STEAP3-AS1 in CRC cell lines and tumor-bearing mouse and zebrafish models under hypoxia. ChIP-qRT-PCR was used to detect the transcriptional activation of STEAP3-AS1 mediated by HIF-1α. RNA-seq, fluorescent in situ hybridization, RNA pulldown, RNA immunoprecipitation, co-immunoprecipitation, immunofluorescence and immunoblot experiments were used to ascertain the involved mechanisms. Functional assays were performed in both in vitro and in vivo models to investigate the regulatory role of STEAP3-AS1 /STEAP3/Wnt/β-catenin axis in CRC proliferation and metastasis. Results Here, we identified a hypoxia-induced antisense lncRNA STEAP3-AS1 that was highly expressed in clinical CRC tissues and positively correlated with poor prognosis of CRC patients. Upregulation of lncRNA STEAP3-AS1 , which was induced by HIF-1α-mediated transcriptional activation, facilitated the proliferation and metastasis of CRC cells both in vitro and in vivo. Mechanistically, STEAP3-AS1 interacted competitively with the YTH domain-containing family protein 2 (YTHDF2), a N 6 -methyladenosine (m 6 A) reader, leading to the disassociation of YTHDF2 with STEAP3 mRNA. This effect protected STEAP3 mRNA from m 6 A-mediated degradation, enabling the high expression of STEAP3 protein and subsequent production of cellular ferrous iron (Fe 2+ ). Increased Fe 2+ levels elevated Ser 9 phosphorylation of glycogen synthase kinase 3 beta (GSK3β) and inhibited its kinase activity, thus releasing β-catenin for nuclear translocation and subsequent activation of Wnt signaling to support CRC progression. Conclusions Taken together, our study highlights the mechanisms of lncRNA STEAP3-AS1 in facilitating CRC progression involving the STEAP3-AS1 /STEAP3/Wnt/β-catenin axis, which may provide novel diagnostic biomarkers or therapeutic targets to benefit CRC treatment. Graphical abstract Hypoxia-induced HIF-1α transcriptionally upregulates the expression of lncRNA STEAP3-AS1 , which interacts competitively with YTHDF2, thus upregulating mRNA stability of STEAP3 and consequent STEAP3 protein expression. The enhanced STEAP3 expression results in production of cellular ferrous iron (Fe 2+ ), which induces the Ser 9 phosphorylation and inactivation of GSK3β, releasing β-catenin for nuclear translocation and contributing to subsequent activation of Wnt signaling to promote CRC progression.