bims-rimeca 2022-02-20 papers

bims-rimeca

Biomed News

on RNA methylation in cancer

Issue of 2022–02–20
twenty papers selected by
Sk Ramiz Islam, Saha Institute of Nuclear Physics

Methylation recognition protein YTH N6-methyladenosine RNA binding protein 1 (YTHDF1) regulates the proliferation, migration and invasion of osteosarcoma by regulating m6A level of CCR4-NOT transcription complex subunit 7 (CNOT7).
m6A demethylase FTO regulates the apoptosis and inflammation of cardiomyocytes via YAP1 in ischemia-reperfusion injury.
The m6A RNA methyltransferase METTL3/METTL14 promotes leukemogenesis through the mdm2/p53 pathway in acute myeloid leukemia.
Construction and validation of m6 A RNA methylation regulators associated prognostic model for gastrointestinal cancer.
METTL14-mediated m6A modification of circORC5 suppresses gastric cancer progression by regulating miR-30c-2-3p/AKT1S1 axis.
The m6A reader YTHDC2 is essential for escape from KSHV SOX-induced RNA decay.
METTL3 regulates LPS-induced inflammatory response via the NOD1 signaling pathway.
Nanoparticle-Induced m6A RNA Modification: Detection Methods, Mechanisms and Applications.
Structural effects of m6A modification of the Xist A-repeat AUCG tetraloop and its recognition by YTHDC1.
Analysis of m6A Methylation Modification Patterns and Tumor Immune Microenvironment in Breast Cancer.
The N6-Methyladenosine Modification and Its Role in mRNA Metabolism and Gastrointestinal Tract Disease.
RNA-Binding Protein IGF2BP1 Associated With Prognosis and Immunotherapy Response in Lung Adenocarcinoma.
Decreased Expression of Programmed Death Ligand-L1 by Seven in Absentia Homolog 2 in Cholangiocarcinoma Enhances T-Cell-Mediated Antitumor Activity.
Validated Impacts of N6-Methyladenosine Methylated mRNAs on Apoptosis and Angiogenesis in Myocardial Infarction Based on MeRIP-Seq Analysis.
ALKBH5 promotes the progression of infantile hemangioma through regulating the NEAT1/miR-378b/FOSL1 axis.
The Non-N6-Methyladenosine Epitranscriptome Patterns and Characteristics of Tumor Microenvironment Infiltration and Mesenchymal Transition in Glioblastoma.
m6A-Finder: Detecting m6A methylation sites from RNA transcriptomes using physical and statistical properties based features.
N-6-Methyladenosine in Vasoactive microRNAs during Hypoxia; A Novel Role for METTL4.
Aberrant translation regulated by METTL1/WDR4-mediated tRNA N7-methylguanosine modification drives head and neck squamous cell carcinoma progression.
The m6A(m)-independent role of FTO in regulating WNT signaling pathways.

Bioengineered. 2022 Mar;13(3): 5236-5250

Methylation recognition protein YTH N6-methyladenosine RNA binding protein 1 (YTHDF1) regulates the proliferation, migration and invasion of osteosarcoma by regulating m6A level of CCR4-NOT transcription complex subunit 7 (CNOT7).

Kang Wei, Yi Gao, Bin Wang, Yu-Xing Qu.

  N6-methyladenosine (m6A) is one of the most significant modifications in human mRNAs. Emerging evidence indicates that m6A participates in the initiation and development of malignant tumors. Nevertheless, the biological roles and mechanism of m6A in osteosarcoma (OS) remain unclear. The purpose of this study was to investigate the role and mechanism of the methylation recognition protein-YTH N6-methyladenosine RNA binding protein 1 (YTHDF1) in OS. The YTHDF1 expression in OS was detected by qRT-PCR and Western blot assay. M6A quantification was utilized to measure the methylation level of OS. Cell counting kit-8 (CCK8), 5-Ethynyl-2'-deoxyuridine (EdU) assay and transwell experiments were conducted to confirm the biological effects of YTHDF1 on OS cells. The bioinformatics websites and in vitro assays were conducted to analyze the downstream targets of YTHDF1 was upregulated in OS tissues at mRNA and protein level. The results showed that the expression level of YTHDF1 might be closely associated with the poor prognosis for OS patients. Inhibition of YTHDF1 could suppress the proliferation, migration and invasion of the OS cells. Moreover, we found that CCR4-NOT transcription complex subunit 7 (CNOT7) might be the potential target of YTHDF1, which was upregulated in OS tissues. YTHDF1 could recognize the m6A sites of CONT7 and promote its expression in an m6A manner. Moreover, methyltransferase-like 3 (METTL3) could promote the m6A level of CONT7. YTHDF1 was upregulated in OS and could promote cell proliferation, migration and invasion. The METTL3-CONT7-YTHDF1 regulatory axis might be the potential target for the prognosis and therapy of OS.

Keywords:  CCR4-NOT transcription complex subunit 7 (CNOT7); Osteosarcoma; YTH N6-methyladenosine RNA binding protein 1 (YTHDF1); malignant progression; methylated modification

DOI:  https://doi.org/10.1080/21655979.2022.2037381
Bioengineered. 2022 Mar;13(3): 5443-5452

m6A demethylase FTO regulates the apoptosis and inflammation of cardiomyocytes via YAP1 in ischemia-reperfusion injury.

Wei-Liang Ke, Zhi-Wen Huang, Chun-Ling Peng, Yi-Ping Ke.

  Reperfusion therapy after acute myocardial infarction can induce myocardial ischemia-reperfusion injury (IRI). Novel evidence has illustrated that N6-methyladenosine (m6A) modification modulates the myocardial IRI progression. Here, our study focuses on the role of m6A methyltransferase fat mass and obesity-associated protein (FTO) in myocardial ischemia/reoxygenation injury and explores potential regulatory mechanisms. Results discovered that FTO down-expressed in myocardial IRI mice and hypoxia/reoxygenation (H/R)-induced cardiomyocytes. Functionally, FTO overexpression attenuated the H/R-induced apoptosis and inflammation of cardiomyocytes. Mechanistically, methylated RNA immunoprecipitation quantitative polymerase chain reaction (MeRIP-qPCR) assay and RIP assay revealed that Yap1 mRNA acted as the target of FTO in cardiomyocytes. Moreover, FTO uninstalled the methylation of Yap1 mRNA, and enforced the stability of Yap1 mRNA. Taken together, our study reveals the role of FTO in H/R-induced myocardial cell injury via m6A-dependent manner, which may provide a new approach to improve myocardial IRI.

Keywords:  Acute myocardial infarction; FTO; N6-methyladenosine; cardiomyocyte

DOI:  https://doi.org/10.1080/21655979.2022.2030572
J Cancer. 2022 ;13(3): 1019-1030

The m6A RNA methyltransferase METTL3/METTL14 promotes leukemogenesis through the mdm2/p53 pathway in acute myeloid leukemia.

Lina Sang, Xia Wu, Tianyou Yan, Duolan Naren, Xiaoyan Liu, Xue Zheng, Nanchen Zhang, Huifang Wang, Yarong Li, Yuping Gong.

  N6-methyladenosine (m6A) is the most abundant internal modification in mammalian mRNA and recent studies have highlighted the importance of m6A levels in tumor development. In this study, we investigated the expression of methyltransferase-like 3 (METTL3) and 14 (METTL14), components of the RNA m6A methyltransferase complex, in samples from 89 patients with acute myeloid leukemia (AML), and followed the survival of 75 of these patients. Our results show that METTL3 and METTL14 are highly expressed in most of the patients with AML (except those with APL), and high levels of METTL3 and/or METTL14 correlated to shorter survival in the patients. In leukemia cell lines K562 and kasumi-1, both METTL3 and METTL14 promote cell proliferation and cell cycle, and the knockdown of METTL3 and METTL14 inhibits proliferation, and induces apoptosis and differentiation. Notably, the knockdown of METTL3 and METTL14 in K562 cell line leads to several changes in the expression of p53 signal pathway, including the upregulation of p53, cyclin dependent kinase inhibitor 1A (CDKN1A/p21), and downregulation of mdm2. Importantly, the m6A level of mdm2 mRNA was significant lower after knock-down of METTL3 and METTL14 examined by m6A-RIP and mdm2 qPCR assay, and the half-life of mdm2 under actinomycin-D treatment became shorter. Taken together, our study demonstrates that the lower m6A levels of mdm2 mRNA mediated by the knockdown of METTL3 and METTL14 could lead to the low stability of mdm2 mRNA transcripts and low expression of MDM2, in the end, activate p53 signal pathway. Both METTL3 and METTL14 play an oncogenic role in AML by targeting mdm2/p53 signal pathway.

Keywords:  METTL14; METTL3; acute myeloblastic leukemia; hematopoietic stem cell; m6A RNA methylation modification

DOI:  https://doi.org/10.7150/jca.60381
IET Syst Biol. 2022 Feb 17.

Construction and validation of m6 A RNA methylation regulators associated prognostic model for gastrointestinal cancer.

Yandong Miao, Bin Su, Xiaolong Tang, Jiangtao Wang, Wuxia Quan, Yonggang Chen, Denghai Mi.

  N6-methyladenosine (m6 A) RNA methylation is correlated with carcinogenesis and dynamically possessed through the m6 A RNA methylation regulators. This paper aimed to explore 13 m6 A RNA methylation regulators' role in gastrointestinal cancer (GIC) and determine the risk model and prognosis value of m6 A RNA methylation regulators in GIC. We used several bioinformatics methods to identify the differential expression of m6 A RNA methylation regulators in GIC, constructed a prognostic model, and carried out functional enrichment analysis. Eleven of 13 m6 A RNA methylation regulators were differentially expressed in different clinicopathological characteristics of GIC, and m6 A RNA methylation regulators were nearly associated with GIC. We constructed a risk model based on five m6 A RNA methylation regulators (METTL3, FTO, YTHDF1, ZC3H13, and WTAP); the risk score is an independent prognosis biomarker. Moreover, the five m6 A RNA methylation regulators can also forecast the 1-, 3- and 5-year overall survival through a nomogram. Furthermore, four hallmarks of oxidative phosphorylation, glycolysis, fatty acid metabolism, and cholesterol homoeostasis gene sets were significantly enriched in GIC. m6 A RNA methylation regulators were related to the malignant clinicopathological characteristics of GIC and may be used for prognostic stratification and development of therapeutic strategies.

Keywords:  RNA modification; bioinformatic analysis; epigenetics; gastrointestinal carcinoma; m6A methylation

DOI:  https://doi.org/10.1049/syb2.12040
Mol Cancer. 2022 02 14. 21(1): 51

METTL14-mediated m6A modification of circORC5 suppresses gastric cancer progression by regulating miR-30c-2-3p/AKT1S1 axis.

Hui-Ning Fan, Zhao-Yu Chen, Xiao-Yu Chen, Ming Chen, You-Cai Yi, Jin-Shui Zhu, Jing Zhang.

   BACKGROUND: N6-methyladenosine (m6A) RNA methylation and circular RNAs (circRNAs) have been shown to act vital roles in multiple malignancies including gastric cancer (GC). However, there is little knowledge about how m6A modification of circRNAs contributes to GC progression.
METHODS: The association of METTL14 expression with the clinicopathological characteristics and prognosis in patients with GC was assessed by Western blot, Immunohistochemistry and public datasets. In vitro and vivo function experiments were conducted to investigate the role of METTL14 in GC. Furthermore, m6A-circRNA epitranscriptomic microarray was utilized to identify METTL14-mediated m6A modification of circRNAs, which were validated by methylated RNA immunoprecipitation (Me-RIP), RT-qPCR and rescue experiments in GC cells. The sponge of circORC5 with miR-30c-2-3p was confirmed by luciferase gene report and RNA immunoprecipitation assays. The expression, localization and prognosis of circORC5 in GC were evaluated by fluorescence in situ hybridization. The effects of METTL14 and (or) circORC5 on miR-30c-2-3p-mediated AKT1S1 and EIF4B were estimated by RT-qPCR and Western blot analyses.
RESULTS: We found that METTL14 was downregulated in GC tissue samples and its low expression acted as a prognostic factor of poor survival in patients with GC. Ectopic expression of METTL14 markedly repressed growth and invasion of GC cells in vitro and in vivo, whereas knockdown of METTL14 harbored the opposite effects. Mechanically, m6A-circRNA epitranscriptomic microarray and Me-RIP identified circORC5 as the downstream target of METTL14. Silencing of METTL14 reduced the m6A level of circORC5, but increased circORC5 expression. Moreover, circORC5 could sponge miR-30c-2-3p, and reverse METTL14-caused upregulation of miR-30c-2-3p and downregulation of AKT1S1 and EIF4B. In addition, circORC5 possessed a negative correlation with miR-30c-2-3p and indicated a poor survival in GC.
CONCLUSION: Our findings demonstrate that METTL14-mediated m6A modification of circORC5 suppresses gastric cancer progression by regulating miR-30c-2-3p/AKT1S1 axis.

Keywords:  Gastric cancer; METTL14; circORC5; m6A; miR-30c-2-3p

DOI:  https://doi.org/10.1186/s12943-022-01521-z
Proc Natl Acad Sci U S A. 2022 Feb 22. pii: e2116662119. [Epub ahead of print]119(8):

The m6A reader YTHDC2 is essential for escape from KSHV SOX-induced RNA decay.

Daniel Macveigh-Fierro, Angelina Cicerchia, Ashley Cadorette, Vasudha Sharma, Mandy Muller.

  The role of N6-methyladenosine (m6A) modifications has increasingly been associated with a diverse set of roles in modulating viruses and influencing the outcomes of viral infection. Here, we report that the landscape of m6A deposition is drastically shifted during Kaposi's sarcoma-associated herpesvirus (KSHV) lytic infection for both viral and host transcripts. In line with previous reports, we also saw an overall decrease in host methylation in favor of viral messenger RNA (mRNA), along with 5' hypomethylation and 3' hypermethylation. During KSHV lytic infection, a major shift in overall mRNA abundance is driven by the viral endoribonuclease SOX, which induces the decay of greater than 70% of transcripts. Here, we reveal that interlukin-6 (IL-6) mRNA, a well-characterized, SOX-resistant transcript, is m6A modified during lytic infection. Furthermore, we show that this modification falls within the IL-6 SOX resistance element, an RNA element in the IL-6 3' untranslated region (UTR) that was previously shown to be sufficient for protection from SOX cleavage. We show that the presence of this m6A modification is essential to confer SOX resistance to the IL-6 mRNA. We next show that this modification recruits the m6A reader YTHDC2 and found that YTHDC2 is necessary for the escape of the IL-6 transcript. These results shed light on how the host cell has evolved to use RNA modifications to circumvent viral manipulation of RNA fate during KSHV infection.

Keywords:  IL-6; RNA decay; herpesvirus; m6A; m6A readers

DOI:  https://doi.org/10.1073/pnas.2116662119
Cell Signal. 2022 Feb 14. pii: S0898-6568(22)00043-2. [Epub ahead of print] 110283

METTL3 regulates LPS-induced inflammatory response via the NOD1 signaling pathway.

Yongjie Cai, Ruiqing Yu, Yiping Kong, Zhihui Feng, Qiong Xu.

  N6-methyladenosine (m6A) is a prevalent mRNA modification that plays a crucial function in multiple biological processes. Methyltransferase-like 3 (METTL3), an m6A methyltransferase, is essential for the m6A modification. Recently, the effect of METTL3 on the immune response has been reported. However, the effect is unclear, and the results are contradictory. In the present study, the total m6A and the expression of METTL3 decreased in LPS-stimulated macrophages. METTL3 knockdown significantly upregulated expression of proinflammatory cytokines, including TNF-α, IL-6 and NO. RNA sequencing analysis showed that the upregulated genes were enriched in inflammation-related signaling pathways and that the NOD-like receptor signaling pathway might be the target molecules of METTL3. METTL3 depletion resulted in upregulation of the NOD1 pathway without impacting NOD2. Moreover, the increase in proinflammatory cytokines induced by METTL3 knockdown was reversed by blocking the NOD1 pathway using specific inhibitors. Mechanistically, METTL3 knockdown promoted the mRNA expression and stability of NOD1 and RIPK2, and the same results were detected in m6A-binding protein YTHDF1- or YTHDF2-silenced cells. All findings suggested that METTL3 depletion inhibits the degradation of NOD1 and RIPK2 mRNA mediated by YTHDF1 and YTHDF2, which upregulate the NOD1 pathway and subsequently promote the LPS-induced inflammatory response in macrophages.

Keywords:  Inflammation; METTL3; Macrophage; NOD1 signaling pathway; mRNA degradation

DOI:  https://doi.org/10.1016/j.cellsig.2022.110283
Nanomaterials (Basel). 2022 Jan 25. pii: 389. [Epub ahead of print]12(3):

Nanoparticle-Induced m6A RNA Modification: Detection Methods, Mechanisms and Applications.

Yi Wang, Fengkai Ruan, Zhenghong Zuo, Chengyong He.

  With the increasing application of nanoparticles (NPs) in medical and consumer applications, it is necessary to ensure their safety. As m6A (N6-methyladenosine) RNA modification is one of the most prevalent RNA modifications involved in many diseases and essential biological processes, the relationship between nanoparticles and m6A RNA modification for the modulation of these events has attracted substantial research interest. However, there is limited knowledge regarding the relationship between nanoparticles and m6A RNA modification, but evidence is beginning to emerge. Therefore, a summary of these aspects from current research on nanoparticle-induced m6A RNA modification is timely and significant. In this review, we highlight the roles of m6A RNA modification in the bioimpacts of nanoparticles and thus elaborate on the mechanisms of nanoparticle-induced m6A RNA modification. We also summarize the dynamic regulation and biofunctions of m6A RNA modification. Moreover, we emphasize recent advances in the application perspective of nanoparticle-induced m6A RNA modification in medication and toxicity of nanoparticles to provide a potential method to facilitate the design of nanoparticles by deliberately tuning m6A RNA modification.

Keywords:  (N6-methyladenosine) RNA modification; epigenetics; function of m6A; nanosafety

DOI:  https://doi.org/10.3390/nano12030389
Nucleic Acids Res. 2022 Feb 15. pii: gkac080. [Epub ahead of print]

Structural effects of m6A modification of the Xist A-repeat AUCG tetraloop and its recognition by YTHDC1.

Alisha N Jones, Ekaterina Tikhaia, André Mourão, Michael Sattler.

The A-repeat region of the lncRNA Xist is critical for X inactivation and harbors several N6-methyladenosine (m6A) modifications. How the m6A modification affects the conformation of the conserved AUCG tetraloop hairpin of the A-repeats and how it can be recognized by the YTHDC1 reader protein is unknown. Here, we report the NMR solution structure of the (m6A)UCG hairpin, which reveals that the m6A base extends 5' stacking of the A-form helical stem, resembling the unmethylated AUCG tetraloop. A crystal structure of YTHDC1 bound to the (m6A)UCG tetraloop shows that the (m6A)UC nucleotides are recognized by the YTH domain of YTHDC1 in a single-stranded conformation. The m6A base inserts into the aromatic cage and the U and C bases interact with a flanking charged surface region, resembling the recognition of single-stranded m6A RNA ligands. Notably, NMR and fluorescence quenching experiments show that the binding requires local unfolding of the upper stem region of the (m6A)UCG hairpin. Our data show that m6A can be readily accommodated in hairpin loop regions, but recognition by YTH readers requires local unfolding of flanking stem regions. This suggests how m6A modifications may regulate lncRNA function by modulating RNA structure.

DOI: https://doi.org/10.1093/nar/gkac080
Front Cell Dev Biol. 2022 ;10 785058

Analysis of m6A Methylation Modification Patterns and Tumor Immune Microenvironment in Breast Cancer.

Menglu Dong, Wenzhuang Shen, Guang Yang, Zhifang Yang, Xingrui Li.

  Increasing evidence indicates that the abnormal expression of N6-methyladenosine (m6A) modification is closely related to the epigenetic regulation of immune response in breast cancer (BC). However, the potential roles of m6A modification in the tumor microenvironment (TME) of BC remain unclear. For addressing this issue, we comprehensively analyzed the m6A modification patterns in 983 samples and correlated these modification patterns with TME immune cell infiltration, based on 23 kinds of m6A regulators. Principal component analysis (PCA) was used to construct the m6A scoring system to quantify the modification pattern of m6A of BC individuals. Consequently, three different m6A modification patterns were identified, and the infiltrating characteristics of TME cells were consistent with the three immune phenotypes, including immune rejection, immune inflammation, and immune desert. Besides, our analysis showed that the prognosis of patients could be predicted by evaluating the m6A modification pattern in the tumor. The low m6Ascore corresponded to increased mutation burden and immune activation, while stroma activation and lack of immune infiltration were observed in high m6Ascore subtypes. In addition, a low m6Ascore was associated with enhanced response to anti-PD-1/PD-L1 immunotherapy. In conclusion, the m6A modification pattern was closely related to the BC immune landscape. This well-validated score model of the m6A modification patterns will provide a valuable tool to depict the tumor immune state and guide effective tumor immunotherapy for combating BC.

Keywords:  M6A; breast cancer; immunotherapy; mutation burden; tumor microenvironment

DOI:  https://doi.org/10.3389/fcell.2022.785058
Front Surg. 2022 ;9 819335

The N6-Methyladenosine Modification and Its Role in mRNA Metabolism and Gastrointestinal Tract Disease.

Teng Cai, Lawrence Lawer Atteh, Xianzhuo Zhang, Chongfei Huang, Mingzhen Bai, Haidong Ma, Chao Zhang, Wenkang Fu, Long Gao, Yanyan Lin, Wenbo Meng.

  The N6-methyladenosine (m6A) modification is the most abundant internal modification of messenger RNA (mRNA) in higher eukaryotes. Under the actions of methyltransferase, demethylase and methyl-binding protein, m6A resulting from RNA methylation becomes dynamic and reversible, similar to that from DNA methylation, and this effect allows the generated mRNA to participate in metabolism processes, such as splicing, transport, translation, and degradation. The most common tumors are those found in the gastrointestinal tract, and research on these tumors has flourished since the discovery of m6A. Overall, further analysis of the mechanism of m6A and its role in tumors may contribute to new ideas for the treatment of tumors. m6A also plays an important role in non-tumor diseases of the gastrointestinal tract. This manuscript reviews the current knowledge of m6A-related proteins, mRNA metabolism and their application in gastrointestinal tract disease.

Keywords:  N6-methyladenosine; gastrointestinal tract; mRNA metabolism; non-tumor diseases; tumor

DOI:  https://doi.org/10.3389/fsurg.2022.819335
Front Genet. 2022 ;13 777399

RNA-Binding Protein IGF2BP1 Associated With Prognosis and Immunotherapy Response in Lung Adenocarcinoma.

JinFeng Liu, Zhi Li, Iokfai Cheang, Jinghang Li, Chunlei Zhou.

  N6-methyladenosine (m6A) is the most common modification in eukaryotic RNAs and plays a vital role in the tumorigenesis and metastasis of various cancers. However, a comprehensive study of m6A methylation regulators in lung adenocarcinoma (LUAD) is still lacking. The present study aimed to systematically explore the role of m6A methylation regulators in LUAD. RNA sequencing data of 20 m6A methylation regulators and clinical data of LUAD patients were downloaded from The Cancer Genome Atlas (TCGA) database. The prognosis value of m6A methylation regulators in LUAD was evaluated using the Gene Expression Profiling Interactive Analysis (GEPIA) and PrognoScan database. The correlation between IGF2BP1 and immune infiltrates in LUAD was investigated via CIBERSORT and Tumor Immune Estimation Resource (TIMER). A total of 15 m6A modification regulators were significantly abnormally expressed in LUAD tissues. Survival analysis revealed that four genes (HNRNPC, HNRNPA2B1, IGF2BP1, and IGF2BP3) were significantly associated with poor prognosis in LUAD. Multivariate Cox regression analysis showed that only IGF2BP1 was an independent predictor of LUAD after adjusting common clinical parameters. The mutation rates of m6A modification regulators in LUAD were less than 10%. Further analysis revealed that IGF2BP1 expression was significantly correlated with immune infiltration, the expression of immune checkpoints, and tumor mutational burden (TMB) in LUAD. Our findings suggest that IGF2BP1 is an independent predictor and related to immunotherapy response in LUAD, which maybe a potential novel biomarker for LUAD prognosis and the status of tumor immunity.

Keywords:  TCGA; immunotherapy response; lung adenocarcinoma; m6A modification regulators; prognosis

DOI:  https://doi.org/10.3389/fgene.2022.777399
Front Immunol. 2022 ;13 845193

Decreased Expression of Programmed Death Ligand-L1 by Seven in Absentia Homolog 2 in Cholangiocarcinoma Enhances T-Cell-Mediated Antitumor Activity.

Hao Zheng, Wen-Juan Zheng, Zhen-Guang Wang, Yuan-Ping Tao, Zhi-Ping Huang, Le Yang, Liu Ouyang, Zhi-Qing Duan, Yi-Nuo Zhang, Bo-Ning Chen, Dai-Min Xiang, Gang Jin, Lu Fang, Fan Zhou, Bo Liang.

  N6-methyladenosine (m6A) has been reported as an important mechanism of post-transcriptional regulation. Programmed death ligand 1 (PD-L1) is a primary immune inhibitory molecule expressed on tumor cells that promotes immune evasion. In addition, seven in absentia homolog 2 (Siah2), a RING E3 ubiquitin ligase, has been involved in tumorigenesis and cancer progression. However, the role of m6A-METTL14-Siah2-PD-L1 axis in immunotherapy remains to be elucidated. In this study, we showed that METTL14, a component of the m6A methyltransferase complex, induced Siah2 expression in cholangiocarcinoma (CCA). METTL14 was shown to enrich m6A modifications in the 3'UTR region of the Siah2 mRNA, thereby promoting its degradation in an YTHDF2-dependent manner. Furthermore, co-immunoprecipitation experiments demonstrated that Siah2 interacted with PD-L1 by promoting its K63-linked ubiquitination. We also observed that in vitro and in vivo Siah2 knockdown inhibited T cells expansion and cytotoxicity by sustaining tumor cell PD-L1 expression. The METTL14-Siah2-PD-L1-regulating axis was further confirmed in human CCA specimens. Analysis of specimens from patients receiving anti-PD1 immunotherapy suggested that tumors with low Siah2 levels were more sensitive to anti-PD1 immunotherapy. Taken together, our results evidenced a new regulatory mechanism of Siah2 by METTL14-induced mRNA epigenetic modification and the potential role of Siah2 in cancer immunotherapy.

Keywords:  METTL14; N6-methyladenosine; PD-L1; Siah2; immunotherapy

DOI:  https://doi.org/10.3389/fimmu.2022.845193
Front Mol Biosci. 2021 ;8 789923

Validated Impacts of N6-Methyladenosine Methylated mRNAs on Apoptosis and Angiogenesis in Myocardial Infarction Based on MeRIP-Seq Analysis.

Yingjie Zhang, Wenjie Hua, Yini Dang, Yihui Cheng, Jiayue Wang, Xiu Zhang, Meiling Teng, Shenrui Wang, Min Zhang, Zihao Kong, Xiao Lu, Yu Zheng.

  Objectives: N6-methyladenosine (m6A) is hypothesized to play a role in the regulation of pathogenesis of myocardial infarction (MI). This study was designed to compare m6A-tagged transcript profiles to identify mRNA-specific changes on pathophysiological variations after MI. Methods: N6-methyladenosine methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA sequencing (RNA-seq) were interacted to select m6A-modified mRNAs with samples collected from sham operated and MI rat models. m6A methylation regulated mRNAs were interacted with apoptosis/angiogenesis related genes in GeneCards. Afterwards, MeRIP-quantitative real-time PCR (MeRIP-qRT-PCR) was performed to measure m6A methylation level of hub mRNAs. m6A methylation variation was tested under different oxygen concentration or hypoxic duration in H9c2 cells and HUVECs. In addition, Western blot and qRT-PCR were employed to detect expression of hub mRNAs and relevant protein level. Flow cytometry and Tunel assay were conducted to assess apoptotic level. CCK-8, EdU, and tube formation assay were performed to measure cell proliferation and tube formation ability. Results: Upregulation of Mettl3 was firstly observed in vivo and in vitro, followed by upregulation of m6A methylation level. A total of 567 significantly changed m6A methylation peaks were identified, including 276 upregulated and 291 downregulated peaks. A total of 576 mRNAs were upregulated and 78 were downregulated. According to combined analysis of MeRIP-seq and RNA-seq, we identified 26 significantly hypermethylated and downregulated mRNAs. Based on qRT-PCR and interactive analysis, Hadh, Kcnn1, and Tet1 were preliminarily identified as hub mRNAs associated with apoptosis/angiogenesis. MeRIP-qRT-PCR assay confirmed the results from MeRIP-seq. With the inhibition of Mettl3 in H9c2 cells and HUVECs, downregulated m6A methylation level of total RNA and upregulated expression of hub mRNAs were observed. Increased m6A level was verified in the gradient context in terms of prolonged hypoxic duration and decreased oxygen concentration. Under simulated hypoxia, roles of Kcnn1 and Tet1 in angiogenesis and Hadh, Tet1, and Kcnn1 in apoptosis were further confirmed with our validation experiments. Conclusion: Roles of m6A-modified mRNA transcripts in the context of MI were preliminarily verified. In the context of m6A methylation, three hub mRNAs were validated to impact the process of apoptosis/angiogenesis. Our study provided theoretical basis and innovative targets for treatment of MI and paved the way for future investigations aiming at exploring upstream epigenetic mechanisms of pathogenesis after MI.

Keywords:  angiogenesis; apoptosis; m6A methylation; mRNA; myocardial infarction

DOI:  https://doi.org/10.3389/fmolb.2021.789923
Mol Cell Biochem. 2022 Feb 18.

ALKBH5 promotes the progression of infantile hemangioma through regulating the NEAT1/miR-378b/FOSL1 axis.

Kun Peng, Ren-Peng Xia, Fan Zhao, Yong Xiao, Ti-Dong Ma, Ming Li, Yong Feng, Chong-Gao Zhou.

  Our work aims to investigate long non-coding RNA (lncRNA) N6-methyladenosine (m6A) modification and its role in infantile hemangioma (IH). The mRNA and protein expression levels were assessed using quantitative real-time polymerase chain reaction, western blot and immunohistochemistry. Me-RIP assay was performed to evaluate lncRNA NEAT1 m6A levels. Cell proliferation, migration and invasion were evaluated using cell counting kit-8 assay, transwell migration and invasion assay, respectively. Photo-activatable ribonucleoside-enhanced crosslinking and immunoprecipitation assay was conducted to verify the binding relationship between lncRNA nuclear paraspeckle assembly transcript 1 (NEAT1) and ALKBH5 (an RNA demethylase). The binding relationship between lncRNA NEAT1, microRNA (miR)-378b and FOS-like antigen 1 (FOSL1) was verified using dual-luciferase reporter gene assay and/or RNA immunoprecipitation assay. ALKBH5, lncRNA NEAT1 and FOLS1 expression was elevated in IH tissues, while miR-378b was downregulated. ALKBH5 knockdown suppressed cell proliferation, migration and invasion of IH cells, while promoting cell apoptosis. ALKBH5 promoted lncRNA NEAT1 expression by reducing the m6A modification of lncRNA NEAT1. In addition, miR-378b was the target of lncRNA NEAT1, and its overexpression reversed the promotion effect of lncRNA NEAT1 overexpression on IH cell tumor-like behaviors. Moreover, FOLS1 was the target of miR-378b, and its overexpression reversed the inhibitory effect of miR-378b overexpression on IH cell tumor-like behaviors in vitro. ALKBH5 might have great potential as therapeutic target for IH, since ALKBH5 silencing suppressed IH progression by regulation of the NEAT1/miR-378b/FOSL1 axis.

Keywords:  ALKBH5; FOSL1; Infantile hemangioma; MiR-378b; lncRNA NEAT1; m6A modification

DOI:  https://doi.org/10.1007/s11010-022-04388-2
Front Immunol. 2021 ;12 809808

The Non-N6-Methyladenosine Epitranscriptome Patterns and Characteristics of Tumor Microenvironment Infiltration and Mesenchymal Transition in Glioblastoma.

Jianye Xu, Zijie Gao, Kaining Liu, Yang Fan, Zongpu Zhang, Hao Xue, Xing Guo, Ping Zhang, Lin Deng, Shaobo Wang, Huizhi Wang, Qingtong Wang, Rongrong Zhao, Gang Li.

   Background: An increasing number of RNA modification types other than N6-methyladenosine (m6A) modification have been detected. Nonetheless, the probable functions of RNA modifications beyond m6A in the tumor microenvironment (TME), mesenchymal (MES) transition, immunotherapy, and drug sensitivity remain unclear.
Methods: We analyzed the characteristics of 32 non-m6A RNA modification regulators in 539 glioblastoma (GBM) patients and the TME cell infiltration and MES transition patterns. Using principal component analysis, a non-m6A epitranscriptome regulator score (RM score) model was established. We estimated the association between RM score and clinical characteristics, TME status, GBM subtypes, and drug and immunotherapy response.
Results: Three definite non-m6A RNA modification patterns associated with diverse biological pathways and clinical characteristics were identified. The high RM score group was characterized by a poor prognosis, enhanced immune infiltration, and MES subtype. Further analysis indicated that the high RM score group had a lower tumor mutation burden as well as a weaker response to immunotherapy. The higher RM score group may benefit more from drugs targeting the EGFR and WNT signaling pathways.
Conclusion: Our study exposed the potential relationship of non-m6A RNA modification regulators with clinical features, TME status, and GBM subtype and clarified its therapeutic value.

Keywords:  glioblastoma; immunotherapy; mesenchymal transition; non-m6A RNA modification; tumor microenvironment

DOI:  https://doi.org/10.3389/fimmu.2021.809808
Comput Biol Chem. 2022 Feb 10. pii: S1476-9271(22)00020-2. [Epub ahead of print]97 107640

m6A-Finder: Detecting m6A methylation sites from RNA transcriptomes using physical and statistical properties based features.

Asad Khan, Hafeez Ur Rehman, Usman Habib, Umer Ijaz.

  N6-methyladenosine (m6A) is one of the abundant post-transcription modification in cellular RNA. It regulates different biological processes, such as, protein synthesis, X-chromosome inactivation, cell stability, cell-reprogramming and miRNA regulation etc. Most recently, various studies claimed that mutations in m6A sites are linked with various diseases, such as, brain-tumor, heart attack, obesity and cancer. The correct identification of m6A sites is essential to overcome these diseases. However, the state-of-the-art predictors face many challenges for precise detection of m6A sites. Even for model organisms, such as Saccharomyces cerevisiae, the detection of m6A sites is difficult due to complex patterns surrounding the m6A sites. These patterns are not widely understood and lead to non-discriminative features for detecting m6A sites. To overcome this problem, we propose a novel predictor called m6A-Finder that creates features based on global and local sequence order. The global sequence order is captured by physical properties based features, while the local sequence order is captured by the statistical features. The fusion of these features results in high dimensional vector which lead to over-fitting, to solve this problem, we use mRMR algorithm to remove redundant features. The proposed technique is evaluated on the most widely used Saccharomyces cerevisiae species dataset. Overall, the m6A-Finder achieved an accuracy of 82.02%, the sensitivity of 82.10%, specificity of 81.94% and a Matthew correlation coefficient value of +0.64.

Keywords:  Feature selection; M6A modification sites; M6A-Finder; MRMR; RNA; SVM

DOI:  https://doi.org/10.1016/j.compbiolchem.2022.107640
Int J Mol Sci. 2022 Jan 19. pii: 1057. [Epub ahead of print]23(3):

N-6-Methyladenosine in Vasoactive microRNAs during Hypoxia; A Novel Role for METTL4.

Daphne A L van den Homberg, Reginald V C T van der Kwast, Paul H A Quax, A Yaël Nossent.

  N-6-methyladenosine (m6A) is the most prevalent post-transcriptional RNA modification in eukaryotic cells. The modification is reversible and can be dynamically regulated by writer and eraser enzymes. Alteration in the levels of these enzymes can lead to changes in mRNA stability, alternative splicing or microRNA processing, depending on the m6A-binding proteins. Dynamic regulation of mRNA m6A methylation after ischemia and hypoxia influences mRNA stability, alternative splicing and translation, contributing to heart failure. In this study, we studied vasoactive microRNA m6A methylation in fibroblasts and examined the effect of hypoxia on microRNAs methylation using m6A immunoprecipitation. Of the 19 microRNAs investigated, at least 16 contained m6A in both primary human fibroblasts and a human fibroblast cell line, suggesting vasoactive microRNAs are commonly m6A methylated in fibroblasts. More importantly, we found that mature microRNA m6A levels increased upon subjecting cells to hypoxia. By silencing different m6A writer and eraser enzymes followed by m6A immunoprecipitation, we identified METTL4, an snRNA m6A methyltransferase, to be predominantly responsible for the increase in m6A modification. Moreover, by using m6A-methylated microRNA mimics, we found that microRNA m6A directly affects downstream target mRNA repression efficacy. Our findings highlight the regulatory potential of the emerging field of microRNA modifications.

Keywords:  METTL3; METTL4; N-6-methyladenosine; fibroblasts; hypoxia; ischemia; m6A; microRNAs; vascular

DOI:  https://doi.org/10.3390/ijms23031057
Cancer Commun (Lond). 2022 Feb 18.

Aberrant translation regulated by METTL1/WDR4-mediated tRNA N7-methylguanosine modification drives head and neck squamous cell carcinoma progression.

Jie Chen, Kang Li, Jianwen Chen, Xiaochen Wang, Rongsong Ling, Maosheng Cheng, Zhi Chen, Fangfang Chen, Qianting He, Shuai Li, Caihua Zhang, Yizhou Jiang, Qianming Chen, Anxun Wang, Demeng Chen.

   BACKGROUND: Cancer cells selectively promote the translation of oncogenic transcripts to stimulate cancer progression. Although growing evidence has revealed that tRNA modifications and related genes participate in this process, their roles in head and neck squamous cell carcinoma (HNSCC) remain largely uncharacterized. Here, we sought to investigate the function and mechanisms of the transfer RNA (tRNA) N7-methylguanosine (m7 G) modification in regulating the occurrence and development of HNSCC.
METHODS: Cell lost-of-function and gain-of-function assays, xenograft models, conditional knockout and knockin mouse models were used to study the physiological functions of tRNA m7 G modification in HNSCC tumorigenesis. tRNA modification and expression profiling, mRNA translation profiling and rescue assays were performed to uncover the underlying molecular mechanisms. Single-cell RNA sequencing (scRNA-seq) was conducted to explore the tumor microenvironment changes.
RESULTS: The tRNA m7 G methyltransferase complex components Methyltransferase-like 1 (METTL1)/WD repeat domain 4 (WDR4) were upregulated in HNSCC and associated with a poor prognosis. Functionally, METTL1/WDR4 promoted HNSCC progression and metastasis in cell-based and transgenic mouse models. Mechanistically, ablation of METTL1 reduced the m7 G levels of 16 tRNAs, inhibiting the translation of a subset of oncogenic transcripts, including genes related to the phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) signaling pathway. In addition, chemical modulators of the PI3K/Akt/mTOR signaling pathway reversed the effects of Mettl1 in mouse HNSCC. Furthermore, scRNA-seq results revealed that Mettl1 knockout in mouse tumor cells altered the immune landscape and cell-cell interaction between the tumor and stromal compartment.
CONCLUSIONS: The tRNA m7 G methyltransferase METTL1 was found to promote the development and malignancy of HNSCC through regulating global mRNA translation, including the PI3K/AKT/mTOR signaling pathway, and found to alter immune landscape. METTL1 could be a promising treatment target for HNSCC patients.

Keywords:  METTL1; PI3K/AKT/mTOR signaling; WDR4; head and neck squamous cell carcinoma; m7G modification; metastasis; microenvironment; scRNA-seq; tRNA

DOI:  https://doi.org/10.1002/cac2.12273
Life Sci Alliance. 2022 05;pii: e202101250. [Epub ahead of print]5(5):

The m6A(m)-independent role of FTO in regulating WNT signaling pathways.

Hyunjoon Kim, Soohyun Jang, Young-Suk Lee.

FTO and ALKBH5 are the two enzymes responsible for mRNA demethylation. Hence, the functional study of FTO has been focused on its mechanistic role in dynamic mRNA modification, and how this post-transcriptional regulation modulates signaling pathways. Here, we report that the functional landscape of FTO is largely associated with WNT signaling pathways but in a manner that is independent of its enzymatic activity. Re-analyses of public datasets identified the bifurcation of canonical and noncanonical WNT pathways as the major role of FTO. In FTO-depleted cells, we find that the canonical WNT/β-Catenin signaling is attenuated in a non-cell autonomous manner via the up-regulation of DKK1. Simultaneously, this up-regulation of DKK1 promotes cell migration via activating the noncanonical WNT/PCP pathway. Unexpectedly, this regulation of DKK1 is independent of its RNA methylation status but operates at the transcriptional level, revealing a noncanonical function of FTO in gene regulation. In conclusion, this study places the functional context of FTO at the branch point of multiple WNT signaling pathways and extends its mechanistic role in gene regulation.

DOI: https://doi.org/10.26508/lsa.202101250