bims-rimeca 2023-01-01 papers

bims-rimeca

Biomed News

on RNA methylation in cancer

Issue of 2023‒01‒01
24 papers selected by
Sk Ramiz Islam
Saha Institute of Nuclear Physics

m6A RNA methylation regulators predict prognosis and indicate characteristics of tumour microenvironment infiltration in acute myeloid leukaemia.
METTL14 Regulates Intestine Cellular Senescence through m6A Modification of Lamin B Receptor.
lncRNA ZNRD1-AS1 promotes malignant lung cell proliferation, migration, and angiogenesis via the miR-942/TNS1 axis and is positively regulated by the m6A reader YTHDC2.
Up-Regulation of SH3TC2 Induced by YTHDF1 Predicts Poor Outcome and Facilitates Cell-Cycle Progress in Colorectal Cancer.
Peptides targeting RNA m6A methylations influence the viability of cancer cells.
A study of RNA m6A demethylases in oral epithelial dysplasia and oral squamous cell carcinoma.
m6A RNA methylation regulator-based signature for prognostic prediction and its potential immunological role in uterine corpus endometrial carcinoma.
RNA N6-methyladenosine modification mediates downregulation of NR4A1 to facilitate malignancy of cervical cancer.
A positive feedback circuit between RN7SK snRNA and m6A readers is essential for tumorigenesis.
ALKBH5 attenuates mitochondrial fission and ameliorates liver fibrosis by reducing Drp1 methylation.
m6A regulator-mediated methylation modification patterns and immune microenvironment infiltration characterization in osteoarthritis.
Molecular Characterization and Clinical Characteristics of m5C-Based RNA Methylation in Spinal Cord Injury: Validated by qPCR.
m6A-related bioinformatics analysis and functional characterization reveals that METTL3-mediated NPC1L1 mRNA hypermethylation facilitates progression of atherosclerosis via inactivation of the MAPK pathway.
Roles of the m6A methyltransferases METTL3, METTL14, and WTAP in pulmonary tuberculosis.
METTL14 alleviates the development of osteoporosis in ovariectomized mice by upregulating m6A level of SIRT1 mRNA.
Elucidating the Kinetic Mechanism of Human METTL16.
N6-methyladenosine modification of viral RNA and its role during the recognition process of RIG-I-like receptors.
MFAP2, upregulated by m1A methylation, promotes colorectal cancer invasiveness via CLK3.
Decoding m6A RNA methylome identifies PRMT6-regulated lipid transport promoting AML stem cell maintenance.
The Role of N6-Methyladenosine in Inflammatory Diseases.
The m6A reader YTHDC2 promotes SIRT3 expression by reducing the stabilization of KDM5B to improve mitochondrial metabolic reprogramming in diabetic peripheral neuropathy.
[N6-methyladenosine (m6A) binding protein hnRNPA2B1 is highly expressed in a variety of tumors and regulates tumor immune microenvironment].
ALKBH5 promotes PD-L1-mediated immune escape through m6A modification of ZDHHC3 in glioma.
KIAA1429 promotes infantile hemangiomas regression by facilitating the stemness of hemangioma endothelial cells.

Epigenetics. 2022 Dec 25. 1-20

m6A RNA methylation regulators predict prognosis and indicate characteristics of tumour microenvironment infiltration in acute myeloid leukaemia.

Xinai Liao, Ling Chen, Jingru Liu, Haoran Hu, Diyu Hou, Ruolan You, Xiaoting Wang, Huifang Huang.

  Patients with acute myeloid leukaemia (AML) have poor prognoses and low overall survival (OS) rates owing to its heterogeneity and the complexity of its tumour microenvironment (TME). N6-methyladenosine (m6A) modification plays a key role in the initiation and progression of haematopoietic malignancies. However, the underlying function of m6A regulators in AML remains elusive. This study thoroughly analysed the m6A modification features of 177 AML patients based on 22 m6A regulators. Utilizing unsupervised clustering, we determined three distinct m6A modification patterns related to different biological functions, TME cell-infiltrating characteristics and clinical outcomes. Additionally, a risk score was constructed based on six m6A regulators-associated prognostic signatures and was validated as an independent and valuable prognostic factor for AML. Patients with a low-risk score exhibited better survival than those with a high-risk score. Many m6A regulators were aberrantly expressed in AML, among which METTL14, YTHDC2, ZC3H13 and RBM15 were observed to be associated with the OS of AML. In addition, these four m6A regulators were found to be noticeably related to the immune checkpoint inhibitor (ICI) treatments. Finally, we verified the expression levels of these four m6A regulators in AML and healthy samples and three groups of AML patients with different risk categories. Collectively, our study indicates that the m6A modification pattern is involved in TME immune-infiltrating characteristics and prognosis in AML. A better understanding of the m6A modification pattern will help enhance our knowledge of the molecular mechanisms of AML and develop potential prognosis prediction indicators and more effective immunotherapeutic strategies.

Keywords:  AML; immunotherapy; m6A; prognosis; tumour microenvironment

DOI:  https://doi.org/10.1080/15592294.2022.2160134
Oxid Med Cell Longev. 2022 ;2022 9096436

METTL14 Regulates Intestine Cellular Senescence through m6A Modification of Lamin B Receptor.

Zizhen Zhang, Meng Xue, Jingyu Chen, Zhuo Wang, Fangyu Ju, Jiaojiao Ni, Jiawei Sun, Haoyue Wu, Huimei Zheng, Ziwei Lou, Yawen Zhang, Xiaohang Yang, Shujie Chen, Yongmei Xi, Liangjing Wang.

N-6-Methyladenosine (m6A) modification is involved in multiple biological processes including aging. However, the regulation of m6A methyltransferase-like 14 (METTL14) in aging remains unclear. Here, we revealed that the level of m6A modification and the expression of METTL14 were particularly decreased in the intestine of aged mice as compared to young mice. Similar results were confirmed in Drosophila melanogaster. Knockdown of Mettl14 in Drosophila resulted in a short lifespan, associated disrupted intestinal integrity, and reduced climbing ability. In human CCD-18Co cells, knockdown of METTL14 accelerated cellular senescence, and the overexpression of METTL14 rescued senescent phenotypes. We also identified the lamin B receptor (LBR) as a target gene for METTL14-mediated m6A modification. Knockdown of METTL14 decreased m6A level of LBR, resulted in LBR mRNA instability, and thus induced cellular senescence. Our findings suggest that METTL14 plays an essential role in the m6A modification-dependent aging process via the regulation of LBR and provides a potential target for cellular senescence.

DOI: https://doi.org/10.1155/2022/9096436
Mol Cancer. 2022 Dec 30. 21(1): 229

lncRNA ZNRD1-AS1 promotes malignant lung cell proliferation, migration, and angiogenesis via the miR-942/TNS1 axis and is positively regulated by the m6A reader YTHDC2.

Jin Wang, Lirong Tan, Xueting Yu, Xiyuan Cao, Beibei Jia, Rui Chen, Jianxiang Li.

  RATIONALE: Lung cancer is the most prevalent form of cancer and has a high mortality rate, making it a global public health concern. The N6-methyladenosine (m6A) modification is a highly dynamic and reversible process that is involved in a variety of essential biological processes. Using in vitro, in vivo, and multi-omics bioinformatics, the present study aims to determine the function and regulatory mechanisms of the long non-coding (lnc)RNA zinc ribbon domain-containing 1-antisense 1 (ZNRD1-AS1).METHODS: The RNAs that were bound to the m6A 'reader' were identified using YTH domain-containing 2 (YTHDC2) RNA immunoprecipitation (RIP)-sequencing. Utilizing methylated RIP PCR/quantitative PCR, pull-down, and RNA stability assays, m6A modification and ZNRD1-AS1 regulation were analyzed. Using bioinformatics, the expression levels and clinical significance of ZNRD1-AS1 in lung cancer were evaluated. Using fluorescent in situ hybridization and quantitative PCR assays, the subcellular location of ZNRD1-AS1 was determined. Using cell migration, proliferation, and angiogenesis assays, the biological function of ZNRD1-AS1 in lung cancer was determined. In addition, the tumor suppressor effect of ZNRD1-AS1 in vivo was validated using a xenograft animal model. Through bioinformatics analysis and in vitro assays, the downstream microRNAs (miRs) and competing endogenous RNAs were also predicted and validated.
RESULTS: This study provided evidence that m6A modification mediates YTHDC2-mediated downregulation of ZNRD1-AS1 in lung cancer and cigarette smoke-exposed cells. Low levels of ZNRD1-AS1 expression were linked to adverse clinicopathological characteristics, immune infiltration, and prognosis. ZNRD1-AS1 overexpression was shown to suppress lung cancer cell proliferation, migration, and angiogenesis in vitro and in vivo, and to reduce tumor growth in nude mice. ZNRD1-AS1 expression was shown to be controlled by treatment of cells with either the methylation inhibitor 3-Deazaadenosine or the demethylation inhibitor Meclofenamic. Furthermore, the miR-942/tensin 1 (TNS1) axis was demonstrated to be the downstream regulatory signaling pathway of ZNRD1-AS1.
CONCLUSIONS: ZNRD1-AS1 serves an important function and has clinical relevance in lung cancer. In addition, the findings suggested that m6A modification could mediate the regulation of the ZNRD1-AS1/miR-942/TNS1 axis via the m6A reader YTHDC2.

Keywords:  Cigarette smoke; Immune infiltration; Lung cancer; TNS1; lncRNA ZNRD1-AS1; m6A RNA methylation; miR-942

DOI:  https://doi.org/10.1186/s12943-022-01705-7
J Oncol. 2022 ;2022 1600611

Up-Regulation of SH3TC2 Induced by YTHDF1 Predicts Poor Outcome and Facilitates Cell-Cycle Progress in Colorectal Cancer.

Huili Wu, Feifei Chu, Lu Li, Kunkun Li, Xingguo Xiao, Li Zhang, Yong Zhang.

N6-methyladenosine (m6A) modification plays a crucial role in determining the fate and function of RNA transcripts in tumor cells. Nevertheless, how m6A regulates the expression of key molecules and coordinates its involvement in the development of colorectal cancer (CRC) remains largely unclear. Here, we report that the m6A reading protein YTHDF1-mediated up-regulation of SH3TC2 promotes CRC growth both in vitro and in vivo. In a pan-cancer analysis across more than thirty types of cancer, we found that SH3TC2 was dysregulated in nine cancers, including BLCA, CHOL, COAD, LAML, PAAD, READ, SKCM, BRCA, and TGCT, and was closely associated with patient prognosis in four cancers, including COAD, MESO, PAAD, and READ. In particular, SH3TC2 was overexpressed in CRC as confirmed by six independent study cohorts. Clinically, high expression of SH3TC2 predicted worse disease-free survival (DFS) in CRC patients. SH3TC2 showed fascinating diagnostic value and was correlated with immunosuppression in CRC. Functionally, RNA-sequencing combined with experiments revealed that knockdown of SH3TC3 significantly inhibited cell-cycle progress of CRC, impairing cell growth. Mechanistically, YTHDF1 protein directly binds with SH3TC2 mRNA and promotes its elevation in an m6A-dependent manner. Thus, our findings provide a mechanism to target the YTHDF1/SH3TC2 axis for CRC therapy.

DOI: https://doi.org/10.1155/2022/1600611
ChemMedChem. 2022 Dec 25.

Peptides targeting RNA m6A methylations influence the viability of cancer cells.

Rushdhi Rauff, Sudeshi Abedeera, Stefani Schmocker, Jiale Xie, Sanjaya Abeysirigunawardena.

  N6-methyladenosine (m 6 A) is the most abundant nucleotide modification observed in eukaryotic mRNA. Changes in m 6 A levels in transcriptome are tightly correlated to expression levels of m 6 A methyltransferases and demethylases. Abnormal expression levels of methyltransferases and demethylases are observed in various diseases and health conditions such as cancer, male infertility, and obesity. This research explores the efficacy of m 6 A-modified RNA as an anticancer drug target. We discovered a 12-mer peptide that binds specifically to m6A-modified RNA using phage display experiments. Our fluorescence-based assays illustrate the selected peptide binds to methylated RNA with lower micromolar affinity and inhibit the binding of protein FTO, a demethylase enzyme specific to m 6 A modification. When cancer cell lines were treated with mtp1, it led to an increase in m 6 A levels and a decrease in cell viability. Hence our results illustrate the potential of mtp1 to be developed as a drug for cancer.

Keywords:  FTO; Phage display; epitranscriptomics; m6A; peptide inhibitors

DOI:  https://doi.org/10.1002/cmdc.202200549
J Oral Biol Craniofac Res. 2023 Mar-Apr;13(2):13(2): 111-116

A study of RNA m6A demethylases in oral epithelial dysplasia and oral squamous cell carcinoma.

Chatchaphan Udompatanakorn, Pichamon Sriviriyakul, Patrayu Taebunpakul.

  Purpose: N6-Methyladenosine (m6A) modification is involved in the tumorigenesis of various cancers. However, the roles of RNA m6A demethylases, fat mass and obesity-associated protein (FTO), and AlkB homolog 5 (ALKBH5) in oral epithelial dysplasia (OED) and oral squamous cell carcinoma (OSCC) remain unclear. This study focuses on FTO and ALKBH5 expression by using immunohistochemistry.Material and methods: Twenty specimens each of OED, OSCC, and normal oral mucosa (NOM) were included. The expression pattern, the number of positive cells, the cell-staining intensity, and the histochemical scoring (H-score) were examined and analyzed.
Results: In all the OED and OSCC specimens, FTO and ALKBH5 were mainly expressed with moderate to strong staining intensity in the nuclei of the abnormal epithelial cells, respectively. Regarding the NOM, both RNA demethylases showed mild cell staining intensity and was present in 50-60% of the specimens. Interestingly, the percentage of cell positivity, the cell-staining intensity, and the H-score of the FTO and ALKBH5 in NOM, OED, and OSCC were increased, respectively (p < 0.001). There was also a positive correlation between the FTO and ALKBH5 expressions in OSCC (r = 0.62, p = 0.003), but not in the NOM and OED.
Conclusion: These results suggest a possible prognostic role of FTO and ALKBH5 expression in the malignant transformation of OED and tumor progression. Further studies are needed to elucidate the mechanisms underlying the roles of FTO and ALKBH5 in carcinogenesis.

Keywords:  ALKBH5; FTO; Oral epithelial dysplasia; Oral precancerous lesions; Oral squamous cell carcinoma; RNA demethylase

DOI:  https://doi.org/10.1016/j.jobcr.2022.12.003
BMC Cancer. 2022 Dec 29. 22(1): 1364

m6A RNA methylation regulator-based signature for prognostic prediction and its potential immunological role in uterine corpus endometrial carcinoma.

Fang Fang, Peipei Wang, Haoyu Huang, Mingquan Ye, Xiaocen Liu, Qingqing Li.

  BACKGROUND: Uterine corpus endometrial carcinoma (UCEC) is the most common female pelvic malignancy worldwide. N6-methyladenosine (m6A) plays an important role in various cellular responses, especially in cancer progression. However, the correlation between prognostic UCEC and m6A RNA methylation regulators remains unclear.METHODS: We used The Cancer Genome Atlas (TCGA) to provide a gene signature that could improve the prognostic evaluation of UCEC patients according to the distinct genetic trait of m6A RNA methylation regulators from a bioinformatics perspective. After comparing UCEC subgroups with different genetic profiles of m6A regulators, we identified 71 differentially expressed genes associated with overall survival (OS) and generated a nine-gene signature through least absolute shrinkage and selection operator (LASSO) Cox regression analysis. Finally, we used in vitro and in vivo tumor cell experiments as well as the immune correlation analysis to verify the function of each gene in the proposed gene signature.
RESULTS: Time-dependent receiver operating characteristic (ROC) curves revealed that the proposed gene signature could predict the outcome of UCEC patients accurately. We found that CDKN2A mainly acted from the perspective of tumor cells, while COL4A4, PXDN, TIGIT, CHODL, LMO3, KCNJ12, L1CAM, and EPHB1 might play a role in UCEC from an immunological point of view.
CONCLUSIONS: From an epigenetics perspective, the m6A RNA methylation regulator-based gene signature can predict the prognosis of UCEC patients and immune therapeutic efficacy.

Keywords:  Gene signature; Prognostic prediction; UCEC; m6A RNA methlation

DOI:  https://doi.org/10.1186/s12885-022-10490-x
Cell Biosci. 2022 Dec 25. 12(1): 207

RNA N6-methyladenosine modification mediates downregulation of NR4A1 to facilitate malignancy of cervical cancer.

Tao Yu, Fuxia Wu, Yan Jia, Xue Zhang, Xiaozhen Qi, Zeyuan Jin, Tongxin Hao, Jianing Zhao, Ziyu Liu, Chaokun Wang, Minmin Niu, Qin Yue, Min Li, Yankun Liu.

  BACKGROUND: N6-methyladenosine is the most abundant eukaryotic mRNA modification and alters a wide range of cellular processes in cancer. Therefore, defining the molecular details are critical for understanding the regulatory mechanism of m6A modification.RESULTS: We found that METTL3, a core m6A methyltransferase component, is upregulated and functions as an oncogene in cervical cancer. Mechanistically, METTL3 induces the degradation of m6A-modified transcripts of NR4A1 though YTHDF2-DDX6 pathway. In addition, NR4A1 overexpression attenuates the malignant progression through recruiting the LSD1/HDAC1/CoREST transcriptional repression complex to AKT1 promoter.
CONCLUSIONS: Our findings reveal that m6A regulates cervical cancer cellular progression through manipulating NR4A1 pathway.

Keywords:  AKT1; Cervical cancer; METTL3; NR4A1; RNA N6-methyladenosine; YTHDF2

DOI:  https://doi.org/10.1186/s13578-022-00937-w
Mol Ther. 2022 Dec 24. pii: S1525-0016(22)00717-1. [Epub ahead of print]

A positive feedback circuit between RN7SK snRNA and m6A readers is essential for tumorigenesis.

Xin Xu, Lifang Ma, Xiao Zhang, Susu Guo, Wanxin Guo, Yikun Wang, Shiyu Qiu, Xiaoting Tian, Yayou Miao, Yongchun Yu, Jiayi Wang.

N6-methyladenosine (m6A) RNA modification, methylation at the N6 position of adenosine, plays critical roles in tumorigenesis. m6A readers recognize m6A modifications and thus act as key executors for the biological consequences of RNA methylation. However, knowledge about the regulatory mechanism(s) of m6A readers is extremely limited. In this study, RN7SK was identified as a small nuclear RNA that interacts with m6A readers. m6A readers recognized and facilitated secondary structure formation of m6A-modified RN7SK, which in turn prevented m6A reader mRNA degradation from exonucleases. Thus, a positive feedback circuit between RN7SK and m6A readers is established in tumor cells. From findings on the interaction with RN7SK, new m6A readers, such as EWS RNA binding protein 1 (EWSR1) and KH RNA binding domain containing, signal transduction associated 1 (KHDRBS1), were identified and shown to boost Wnt/βcatenin signaling and tumorigenesis by suppressing translation of Cullin1 (CUL1). Moreover, several Food and Drug Administration-approved small molecules were demonstrated to reduce RN7SK expression and inhibit tumorigenesis. Together, these findings reveal a common regulatory mechanism of m6A readers and indicate that targeting RN7SK has strong potential for tumor treatment.

DOI: https://doi.org/10.1016/j.ymthe.2022.12.013
Pharmacol Res. 2022 Dec 21. pii: S1043-6618(22)00554-0. [Epub ahead of print]187 106608

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

Juan Wang, Yang Yang, Feng Sun, Yong Luo, Yan Yang, Jun Li, Wei Hu, Hui Tao, Chao Lu, Jing-Jing Yang.

  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.

Keywords:  ALKBH5; Drp1; Hepatic stellate cells; Liver fibrosis; Mitochondrial fission; YTHDF1

DOI:  https://doi.org/10.1016/j.phrs.2022.106608
BMC Med Genomics. 2022 Dec 30. 15(1): 273

m6A regulator-mediated methylation modification patterns and immune microenvironment infiltration characterization in osteoarthritis.

Shidong Hu, Chen Shen, Xudong Yao, Yulong Zou, Ting Wang, Xianding Sun, Mao Nie.

  Osteoarthritis (OA) is a common disease in orthopedics. RNA N6-methyladenosine (m6A) exerts an essential effect in a variety of biological processes in the eukaryotes. In this study, we determined the effect of m6A regulators in the OA along with performing the subtype classification. Differential analysis of OA and normal samples in the database of Gene Expression Omnibus identified 9 significantly differentially expressed m6A regulators. These regulators were monitored by a random forest algorithm so as to evaluate the risk of developing OA disease. On the basis of these 9 moderators, a nomogram was established. The results of decision curve analysis suggested that the patients could benefit from a nomogram model. The OA sample was classified as 2 m6A models through a consensus clustering algorithm in accordance with these 9 regulators. These 2 m6A patterns were then assessed with principal component analysis. We also determined the m6A scores for the 2 m6A patterns and their correlation with immune infiltration. The results indicated that type A had a higher m6A score than type B. Thus, we suggest that the m6A pattern may provide a new approach for diagnose and provide novel ideas for molecular targeted therapy of OA.

Keywords:  Bioinformatic analysis; Immune infiltration; Osteoarthritis; RNA N6-methyladenosine; Subtype classification

DOI:  https://doi.org/10.1186/s12920-022-01429-z
Int J Genomics. 2022 ;2022 5433860

Molecular Characterization and Clinical Characteristics of m5C-Based RNA Methylation in Spinal Cord Injury: Validated by qPCR.

Liang Cao, Wen Jun Pi, Qiang Zhang, Qing Li.

Aberrant patterns of 5-methylcytosine (m5C)-based ribonucleic acid (RNA) methylation have critical roles in various human diseases, but their importance in spinal cord injury (SCI) is largely unknown. We explore the expression patterns and potential roles of m5C-based regulators of RNA modification after SCI. We analyzed 16 m5C-based regulators of RNA modification in tissues with SCI and normal rats from the Gene Expression Omnibus database. We constructed a "gene signature" of m5C-based regulators of RNA modification to predict the prognosis of SCI using least absolute shrinkage and selection operator regression and random-forest strategy. We found that the m5C-related genes, deoxyribonucleic acid (DNA) methyltransferase1 (Dnmt1), methyl-CpG binding domain protein 2 (Mbd2), ubiquitin-like with PHD and ring finger domains 1 (Uhrf1), uracil-N-glycosylase (Ung), and zinc finger and BTB(brica-brac, tramtrack, and broad) domain containing 38 (Zbtb38) had high expression, and zinc finger and BTB domain containing 4 (Zbtb4) had low expression in SCI. Analysis of the correlation between the gene sets of m5C-based regulators of RNA modification and immune-cell infiltration and immune response revealed Dnmt1, DNA methyltransferases 3A (Dnmt3a), Mbd2, and Ung to be positive regulators of the immune microenvironment, and Zbtb4 may negatively regulate the immune environment. Then, two molecular subtypes were identified based on 16 m5C-regulated genes. Functional-enrichment analysis of differentially expressed genes between different patterns of m5C-based modification was undertaken. Through the creation of a protein-protein interaction network, we screened 11 hub genes. We demonstrated their importance between SCI group and sham group using real-time reverse transcription-quantitative polymerase chain reaction in rat model. Expression of hub genes did not correlate with mitophagy but was positively correlated with endoplasmic reticulum stress (ERS), which suggested that there may be differences in ERS between different patterns of m5C-based modification. This present study explored and discovered the close link between m5C regulators-related genes and SCI. We also hope our findings may contribute to further mechanistic and therapeutic research on the role of key m5C regulators after SCI.

DOI: https://doi.org/10.1155/2022/5433860
Inflamm Res. 2022 Dec 30.

m6A-related bioinformatics analysis and functional characterization reveals that METTL3-mediated NPC1L1 mRNA hypermethylation facilitates progression of atherosclerosis via inactivation of the MAPK pathway.

Guoan Zhang, Xuewen Li, Xiaoyan Huang.

  OBJECTIVE: Accumulating evidence has demonstrated that N6-methyladenosine (m6A) plays important roles in many major diseases, including atherosclerosis (AS). In the present study, we aimed to explore the transcriptomic m6A landscape of endothelial function-associated genes and identify potential regulators in AS progression.METHODS: The GEO data (GSE142386) from MeRIP-seq in human umbilical vein endothelial cells (HUVECs) with METTL3 knocked down or not were analyzed. RNA-seq was performed to identify differences in gene expression. Gene ontology (GO) functional and Kyoto encyclopedia of genes and genomes (KEGG) pathway analyses were conducted to evaluate the potential functions of the differentially expressed genes. MeRIP-qPCR was used to measure the m6A and mRNA levels of the top 8 downregulated genes, and NPC1L1 was selected as the candidate gene. Oxidized low-density lipoprotein (ox-LDL) was used to stimulate HUVECs, and METTL3 or NPC1L1 was silenced in ox-LDL-treated cells. And Transwell, ELISA, and cell apoptosis assays were performed to assess cell functional injury. ApoE-/- mice were fed with high-fat diet for 8 weeks to establish an AS model, and adenovirus-mediated NPC1L1 shRNA or NC shRNA was injected into the mice through the tail vein. Mouse aortic tissue damage and plaque deposition were evaluated by H&E, Oil Red O, and TUNEL staining.
RESULTS: One hundred and ninety-four hypermethylated m6A peaks and 222 hypomethylated peaks were detected in response to knockdown of METTL3. Genes with altered m6A peaks were significantly involved in the histone modification, enzyme activity, and formation of multiple complexes and were predominantly enriched in the MAPK pathway. NPC1L1 was a most significantly downregulated transcript in response to knockdown of METTL3. Moreover, knockdown of NPC1L1 or de-m6A (METTL3 knockdown)-mediated downregulation of NPC1L1 could improve ox-LDL-induced dysfunction of HUVECs in vitro and high-fat diet-induced atherosclerotic plaque in vivo, which was associated with the inactivation of the MAPK pathway.
CONCLUSION: METTL3-mediated NPC1L1 mRNA hypermethylation facilitates AS progression by regulating the MAPK pathway, and NPC1L1 may be a novel target for the treatment of AS.

Keywords:  Atherosclerosis; Human umbilical vein endothelial cells; METTL3; N6-Methyladenosine; NPC1L1

DOI:  https://doi.org/10.1007/s00011-022-01681-0
Front Immunol. 2022 ;13 992628

Roles of the m6A methyltransferases METTL3, METTL14, and WTAP in pulmonary tuberculosis.

Tian-Ping Zhang, Rui Li, Li-Jun Wang, Qian Huang, Hong-Miao Li.

  Objective: The aim of the current study was to investigate the contributing role of gene variation and transcription levels among the m6A methyltransferases METTL3, METTL14, and WTAP in pulmonary tuberculosis (PTB).Methods: A case-control study including 461 PTB patients and 467 normal controls was designed for genotyping. Three SNPs in METTL3 (rs1061027, rs1139130, rs1061026), three SNPs in METTL14 (rs62328061, rs4834698, rs1064034), and two SNPs in WTAP (rs1853259, rs11752345) were genotyped via the SNPscan™ technique. METTL3, METTL14, and WTAP transcription levels were determined in 78 PTB patients and 86 controls via quantitative real-time reverse-transcription PCR.
Results: Frequencies of the METTL14 rs62328061 GG genotype, WTAP rs11752345 CT genotype, and T allele were significantly increased in PTB patients compared to controls. An increased risk of rs62328061 was detected in a recessive model, and a decreased risk of rs11752345 was detected in a dominant model in the PTB group. METTL3 gene variation was not associated with PTB risk. The METTL3 rs1139130 GG genotype was significantly increased with drug resistance, and the G allele was significantly decreased with drug-induced liver injury in PTB patients. A reduced frequency of the METTL14 rs62328061 G allele was associated with leukopenia, a reduced frequency of the WTAP rs11752345 T allele was associated with sputum smear positivity, and a higher frequency of the METTL14 rs4834698 TC genotype was evident in PTB patients with hypoproteinemia. Compared to controls, METTL3, METTL14, and WTAP transcription levels in PTB patients were significantly decreased, and the level of WTAP was increased in PTB patients with drug resistance. METTL3 level was negatively associated with erythrocyte sedimentation rate and aspartate aminotransferase, and METTL14 level was negatively correlated with alanine aminotransferase and aspartate aminotransferase.
Conclusion: METTL14 rs62328061 and WTAP rs11752345 variants were associated with the genetic background of PTB, and METTL3, METTL14, and WTAP levels were abnormally decreased, suggesting that these m6A methyltransferases may play important roles in PTB.

Keywords:  METTL14; METTL3; WTAP; m6A methyltransferase; pulmonary tuberculosis; single nucleotide polymorphisms

DOI:  https://doi.org/10.3389/fimmu.2022.992628
Bone. 2022 Dec 27. pii: S8756-3282(22)00329-5. [Epub ahead of print] 116652

METTL14 alleviates the development of osteoporosis in ovariectomized mice by upregulating m6A level of SIRT1 mRNA.

Changsheng Wang, Rongsheng Chen, Xitian Zhu, Xiaobo Zhang, Nancheng Lian.

  The purpose of this study was to investigate whether METTL14 participated in ovariectomized (OVX)-induced osteoporosis (OP) in mice by regulating the m6A level of SIRT1 mRNA. OVX was performed on mice to induce OP, and mouse bone marrow stromal cells (BMSCs) and bone marrow mononuclear macrophages (BMMs) were isolated to induce osteoblast differentiation and osteoclast differentiation, respectively. The morphology of bone trabeculae was evaluated under a micro-CT scanner. The changes in pathology of bone tissues were observed through staining using hematoxylin-eosin. The number of osteoclasts was measured by tartrate-resistant acid phosphatase staining, and the content of serum calcium, PINP, and CTX-I was tested by enzyme-linked immunosorbent assay, accompanied by the measurement of the expression of SIRT1, METTL14, osteogenic marker genes, and osteoclast marker genes. The m6A modification level of SIRT1 and the binding between METTL14 and SIRT1 were verified. In OVX mice, SIRT1 and METTL14 were downregulated. Overexpression of SIRT1 or METTL14 increased the expression of osteogenic marker genes but decreased the expression of osteoclast marker genes. Additionally, METTL14 overexpression increased m6A level of SIRT1 mRNA. Furthermore, overexpression of METTL14 promoted osteoblast differentiation and suppressed osteoclast differentiation, which were reversed by knockdown of SIRT1. METTL14 promoted osteoblast differentiation and repressed osteoclast differentiation by m6A-dependent upregulation of SIRT1 mRNA, thereby alleviating OP development.

Keywords:  METTL14; Osteoblast differentiation; Osteoclast differentiation; Osteoporosis; SIRT1

DOI:  https://doi.org/10.1016/j.bone.2022.116652
Biochemistry. 2022 Dec 30.

Elucidating the Kinetic Mechanism of Human METTL16.

Kurtis Breger, Jessica A Brown.

Methyltransferase-like protein 16 (METTL16) is one of four catalytically active, S-adenosylmethionine (SAM)-dependent m6A RNA methyltransferases in humans. Well-known methylation targets of METTL16 are U6 small nuclear RNA (U6 snRNA) and the MAT2A mRNA hairpins; however, METTL16 binds to other RNAs, including the 3' triple helix of the metastasis-associated lung adenocarcinoma transcript 1 (MALAT1). Herein, we investigated the kinetic mechanism and biochemical properties of METTL16. METTL16 is a monomer in complex with either the MALAT1 triple helix or U6 snRNA and binds to these RNAs with respective dissociation constants of 31 nM and 18 nM, whereas binding to the methylated U6 snRNA product is 1.1 μM. The MALAT1 triple helix, on the other hand, is not methylated by METTL16 under in vitro conditions. Using the U6 snRNA to study methylation steps, preincubation and isotope partitioning assays indicated an ordered-sequential mechanism, whereby METTL16 binds U6 snRNA before SAM. The apparent dissociation constant for the METTL16·U6 snRNA·SAM ternary complex is 126 μM. Steady-state kinetic assays established a kcat of 0.07 min-1, and single-turnover assays established a kchem of 0.56 min-1. Furthermore, the methyltransferase domain of METTL16 methylated U6 snRNA with an apparent dissociation constant of 736 μM and a kchem of 0.42 min-1, suggesting that the missing vertebrate conserved regions weaken the ternary complex but do not induce any rate-limiting conformational rearrangements of the U6 snRNA. This study helps us to better understand the catalytic activity of METTL16 in the context of its biological functions.

DOI: https://doi.org/10.1021/acs.biochem.2c00601
Front Immunol. 2022 ;13 1031200

N6-methyladenosine modification of viral RNA and its role during the recognition process of RIG-I-like receptors.

Huanan Li, Yang Guo, Wenbao Qi, Ming Liao.

  N6-methyladenosine (m6A) is the most abundant RNA chemical modification in eukaryotes and is also found in the RNAs of many viruses. In recent years, m6A RNA modification has been reported to have a role not only in the replication of numerous viruses but also in the innate immune escape process. In this review, we describe the viruses that contain m6A in their genomes or messenger RNAs (mRNAs), and summarize the effects of m6A on the replication of different viruses. We also discuss how m6A modification helps viral RNAs escape recognition by exogenous RNA sensors, such as retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), during viral invasion. Overall, the goal of our review is to summarize how m6A regulates viral replication and facilitates innate immune escape. Furthermore, we elaborate on the potential of m6A as a novel antiviral target.

Keywords:  RIG-I-like receptors; innate immune escape; m6A; replication; viruses

DOI:  https://doi.org/10.3389/fimmu.2022.1031200
Cancer Med. 2022 Dec 30.

MFAP2, upregulated by m1A methylation, promotes colorectal cancer invasiveness via CLK3.

Meng Xue, Shuyi Mi, Zizhen Zhang, Hao Wang, Wenwen Chen, Wei Wei, Guochun Lou.

  BACKGROUND: Distant metastasis is the main cause of mortality in colorectal cancer (CRC) patients. N1-methyladenosine (m1A) is a type of epitranscriptome modification. While its regulatory effect on mRNA and its role in CRC metastasis remain unclear.METHODS: The m1A methylation profile of mRNAs in CRC was revealed by m1A methylated RNA immunoprecipitation sequencing. The expression of MFAP2 in tumor tissues was measured by immunohistochemistry and then correlated with the clinical characteristics and prognosis of CRC patients. The role of MFAP2 in the invasiveness of CRC cells was evaluated by transwell assays and peritoneal metastatic model in nude mice. The downstream targets of MFAP2 was screened by mass spectrometry analysis. Then the role of MFAP2-CLK3 signaling axis was verified by cotransfecting MFAP2 siRNA and CLK3 plasmid in CRC cells.
RESULTS: Microfibril associated protein 2 (MFAP2) mRNA was overexpressed and m1A-hypermethylated in CRC. High expression of MFAP2 was closely related to lymph node metastasis and distant metastasis, leading to poor prognosis in patients with CRC. In vivo and in vitro studies showed that silencing of MFAP2 inhibited the migration, invasion and metastasis of CRC cells. CDC Like Kinase 3 (CLK3) was a potential downstream target of MFAP2. Further studies showed that MFAP2 depletion might induce autophagic degradation of CLK3, and the role of MFAP2 in the invasiveness of CRC cells was dependent on CLK3.
CONCLUSIONS: Our results uncover a newly identified MFAP2-CLK3 signaling axis, which is a potential therapeutic target for CRC metastasis.

Keywords:  MFAP2; N1-methyladenosine; colorectal cancer; invasiveness

DOI:  https://doi.org/10.1002/cam4.5561
Cell Stem Cell. 2022 Dec 17. pii: S1934-5909(22)00487-8. [Epub ahead of print]

Decoding m6A RNA methylome identifies PRMT6-regulated lipid transport promoting AML stem cell maintenance.

Ying Cheng, Zhuying Gao, Tiantian Zhang, Yuhua Wang, Xueqin Xie, Guoqiang Han, Yashu Li, Rong Yin, Yilin Chen, Peipei Wang, Jin Hu, Tong Zhang, Chengli Guo, Jihua Chai, Jing Wang, Manman Cui, Kexin Gao, Weidong Liu, Shuxin Yao, Pengbo Lu, Ziyan Cao, Yanbing Zheng, Jiwei Chang, Zheming Liu, Qibin Song, Weiming Li, Fuling Zhou, Haojian Zhang.

  N6-methyladenosine (m6A) is a common chemical modification for mammalian mRNA and exhibits high dynamics in various biological processes. However, dynamics of m6A RNA methylome during leukemogenesis remains unknown. Here, we delineate a comprehensive m6A landscape during acute myeloid leukemia (AML) development and identify PRMT6 as a key for maintaining AML stem cells. We observe an obvious change in m6A methylome during leukemogenesis and find that protein arginine methyltransferase PRMT6 and m6A reader IGF2BP2 maintain the function of human and murine leukemia stem cells (LSCs). Genetic deletion or pharmacological inhibition of PRMT6 damages AML development and LSC function. Mechanistically, IGF2BP2 stabilizes PRMT6 mRNA via m6A-mediated manner, which catalyzes H3R2me2a and suppresses lipid transporter MFSD2A expression. PRMT6 loss upregulates MFSD2A expression that increases docosahexaenoic acid levels and impairs LSC maintenance. Collectively, our findings reveal a critical role of PRMT6-MFSD2A signaling axis in AML development and provide a therapeutic strategy for targeting LSCs.

Keywords:  IGF2BP2; PRMT6; RNA m(6)A; acute myeloid leukemia; leukemia stem cells

DOI:  https://doi.org/10.1016/j.stem.2022.12.003
Oxid Med Cell Longev. 2022 ;2022 9744771

The Role of N6-Methyladenosine in Inflammatory Diseases.

Haojun Xu, Changjie Lin, Jinghan Yang, Xi Chen, Yingyu Chen, Jianguo Chen, Aizhen Guo, Changmin Hu.

N6-Methyladenosine (m6A) is the most abundant epigenetic RNA modification in eukaryotes, regulating RNA metabolism (export, stability, translation, and decay) in cells through changes in the activity of writers, erasers, and readers and ultimately affecting human life or disease processes. Inflammation is a response to infection and injury in various diseases and has therefore attracted significant attention. Currently, extensive evidence indicates that m6A plays an essential role in inflammation. In this review, we focus on the mechanisms of m6A in inflammatory autoimmune diseases, metabolic disorder, cardio-cerebrovascular diseases, cancer, and pathogen-induced inflammation, as well as its possible role as targets for clinical diagnosis and treatment.

DOI: https://doi.org/10.1155/2022/9744771
Acta Diabetol. 2022 Dec 27.

The m6A reader YTHDC2 promotes SIRT3 expression by reducing the stabilization of KDM5B to improve mitochondrial metabolic reprogramming in diabetic peripheral neuropathy.

Yang Jiao, Shu Wang, Xin Wang, Ling Yin, Yue-Hua Zhang, Yi-Ze Li, Yong-Hao Yu.

  AIMS: Diabetic peripheral neuropathy (DPN) is a common diabetic complication. Aberrant mitochondrial function causes neurodegeneration under hyperglycemia-induced metabolic stress, which in turn results in DPN progression. m6A and m6A reader (YTHDC2) are closely related to diabetes and diabetes complications, while the role of YTHDC2 in regulating mitochondrial metabolism in DPN needs to be further probed.METHODS: For HG treatment, Schwann cells (RSC96) were subjected to D-glucose for 72 h. db/db mice were used as the diabetic mouse model. Me-RIP assay was performed to evaluate KDM5B m6A level. RNA degradation assay was conducted to examine KDM5B mRNA stability. In addition, OCR and ECAR were examined by XF96 Analyzer. Moreover, the content of ATP and PDH activity in RSC96 cells were detected using kits, and the level of ROS was detected using MitoSOX staining. RIP, RNA pull-down and dual-luciferase reporter gene assays were carried out to verify the binding relationships between YTHDC2, KDM5B and SIRT3.
RESULTS: We first observed that KDM5B expression and KDM5B mRNA stabilization were significantly increased in DPN. The m6A reader YTHDC2 was lowly expressed in DPN. Meanwhile, YTHDC2 over expression decreased KDM5B mRNA stability in an m6A-dependent manner. Our results also revealed that YTHDC2 overexpression resulted in reduced ROS level and increased ATP level, PDH activity, OCR and ECAR in HG-treated Schwann cells, while these effects were reversed by KDM5B overexpression. Additionally, SIRT3 served as the target of YTHDC2/KDM5B axis in regulating mitochondrial metabolism in DPN.
CONCLUSIONS: Taken together, YTHDC2 promoted SIRT3 expression by reducing the stabilization of KDM5B to improve mitochondrial metabolic reprogramming in DPN.

Keywords:  Diabetic peripheral neuropathy; KDM5B; Mitochondrial metabolic reprogramming; SIRT3; YTHDC2

DOI:  https://doi.org/10.1007/s00592-022-01990-0
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2022 Dec;38(12): 1069-1077

[N6-methyladenosine (m6A) binding protein hnRNPA2B1 is highly expressed in a variety of tumors and regulates tumor immune microenvironment].

Jiayao Wang, Xiaodi Zhao, Yuanyuan Lu, Daiming Fan.

Objective To investigate the expression of N6-methyladenosine (m6A) binding protein hnRNPA2B1 in various tumors and its relationship with prognosis and immune infiltration. Methods We investigated the expression of hnRNPA2B1 in different tumors and verified it in gastric cancer (GC) tissue microarray using immunohistochemistry. Univariate COX regression and Kaplan-Meier survival analysis were used to identify the prognostic value of hnRNPA2B1 in pan-cancer. In addition, we explored the correlation between the expression of hnRNPA2B1 and immune cell infiltration, immune checkpoint genes, tumor mutational burden (TMB) as well as microsatellite instability (MSI). Results The expression of hnRNPA2B1 was higher in tumor tissues than in corresponding normal tissues in most cancers. In GC tissue microarray, the expression of hnRNPA2B1 in GC tissues was significantly higher than that in paired adjacent normal tissues. High expression of hnRNPA2B1 was significantly associated with poor prognosis in 7 types of tumors. Moreover, the expression of hnRNPA2B1 was positively correlated with immune cell infiltration in a variety of tumors. In addition, hnRNPA2B1 was notably associated with immune checkpoint related genes, TMB and MSI. Conclusion The expression of hnRNPA2B1 is ubiquitously elevated in a variety of tumors and is associated with poor prognosis. Furthermore, hnRNPA2B1 is closely related to the immune cell infiltration and tumor microenvironment.
Cell Death Discov. 2022 Dec 24. 8(1): 497

ALKBH5 promotes PD-L1-mediated immune escape through m6A modification of ZDHHC3 in glioma.

Wenhui Tang, Ningbo Xu, Jian Zhou, Zhenyan He, Cameron Lenahan, Chenyang Wang, Huangyi Ji, Boyang Liu, Yujiao Zou, Huijun Zeng, Hongbo Guo.

N6-methylation of adenosine (m6A) is one of the most frequent chemical modifications in eukaryotic RNAs and plays a vital role in tumorigenesis and progression. Recently, emerging studies have shown that m6A modification by ALKBH5 was associated with immunotherapy response in various types of cancer. However, whether m6A demethylases ALKBH5 participate in regulating the tumor immune microenvironment and the efficacy of immunotherapy in glioblastoma remain unknown. Here, we found that deletion of ALKBH5 significantly inhibited the growth of glioma allografts, rescued the antitumoral immune response, and increased cytotoxic lymphocyte infiltration and proinflammatory cytokines in CSF while significantly suppressing PD-L1 protein expression. m6A-methylated RNA immunoprecipitation sequencing and RNA sequencing identify ZDDHC3 as the direct target of ALKBH5. Mechanically, ALKBH5 deficiency impairs the YTHDF2-mediated stability of ZDHHC3 mRNA, thereby suppressing PD-L1 expression by accelerating PD-L1 degradation in glioma. In addition, genetic deletion or pharmacological inhibition of ALKBH5 with IOX1 enhances the therapeutic efficacy of anti-PD-1 treatment in preclinical mice models. These data suggest that the combination of anti-PD-1 therapy and ALKBH5 inhibition may be a promising treatment strategy in glioma.

DOI: https://doi.org/10.1038/s41420-022-01286-w
Cancer Sci. 2022 Dec 26.

KIAA1429 promotes infantile hemangiomas regression by facilitating the stemness of hemangioma endothelial cells.

Luying Wang, Yuqing Zou, Zhishun Huang, Wenjing Wang, Jing Li, Jianhai Bi, Ran Huo.

  Infantile hemangiomas are common vascular tumors with specific natural history. The proliferation and regression mechanism of infantile hemangiomas may be related to the multilineage differentiation ability of hemangioma stem cells, but the specific mechanism is not well elucidated. KIAA1429 is an N6 -methyladenosine methylation-related proteins that can also exert its role in a methylation-independent manner. This study aims to explore the function of KIAA1429 in infantile hemangiomas. qRT-PCR, western blotting, and immunostaining were performed to verify the expression of KIAA1429. The endothelial and fibroblast-like phenotype of hemangioma endothelial cells were detected after KIAA1429 knockdown and overexpression. The Stemness properties of hemangioma endothelial cells and the underlying mechanism of KIAA1429 in hemangiomas were also investigated. Nude mouse models of infantile hemangiomas were conducted to ascertain the effects of KIAA1429 in vivo. The results showed that KIAA1429 was highly expressed in infantile hemangiomas, particularly in involuting hemangiomas. In vitro experiments confirmed that KIAA1429 inhibited the endothelial phenotype, enhanced the differentiation ability, and promoted the fibroblast-like phenotype of hemangioma endothelial cells by inducing endothelial cells transition to facultative stem cells. However, the effect of KIAA1429 on the potential target was shown to be independent of N6 -methyladenosine methylation modification. Mouse models further revealed that KIAA1429 could inhibit the proliferation and promote the regression of hemangiomas. In conclusion, this study found that KIAA1429 played an important role in the regression of infantile hemangiomas by enhancing the stemness of hemangioma endothelial cells and could be a potential treatment target for infantile hemangiomas.

Keywords:  cell differentiation; endothelial cells; fibroblast; hemangioma; stem cells

DOI:  https://doi.org/10.1111/cas.15708