bims-rimeca 2022-01-30 papers

bims-rimeca

Biomed News

on RNA methylation in cancer

Issue of 2022–01–30
23 papers selected by
Sk Ramiz Islam, Saha Institute of Nuclear Physics

The essential roles of m6A RNA modification to stimulate ENO1-dependent glycolysis and tumorigenesis in lung adenocarcinoma.
The multifaceted functions of the Fat mass and Obesity-associated protein (FTO) in normal and cancer cells.
Current Advances in N6-Methyladenosine Methylation Modification During Bladder Cancer.
Characterization of m6A Regulator-Mediated Methylation Modification Patterns and Tumor Microenvironment Infiltration in Ovarian Cancer.
β-Elemene Restrains PTEN mRNA Degradation to Restrain the Growth of Lung Cancer Cells via METTL3-Mediated N6 Methyladenosine Modification.
YTHDF2 promotes multiple myeloma cell proliferation via STAT5A/MAP2K2/p-ERK axis.
N6-methyladenosine-induced SVIL antisense RNA 1 restrains lung adenocarcinoma cell proliferation by destabilizing E2F1.
m6A Regulators Mediated Methylation Modification Patterns and Tumor Microenvironment Infiltration Characterization In Nasopharyngeal Carcinoma.
scDART-seq reveals distinct m6A signatures and mRNA methylation heterogeneity in single cells.
Comprehensive Analysis of Differentially Expressed Profiles of mRNA N6-Methyladenosine in Colorectal Cancer.
Prognostic implications of N6-methyladenosine RNA regulators in breast cancer.
The Interaction Between N6-Methyladenosine Modification and Non-Coding RNAs in Gastrointestinal Tract Cancers.
Comprehensive analysis of N6-methylandenosine regulators and m6A-related RNAs as prognosis factors in colorectal cancer.
Mechanism of METTL3-Mediated m6A Modification in Cardiomyocyte Pyroptosis and Myocardial Ischemia-Reperfusion Injury.
Ubiquitous Knockdown of Mettl3 using TRiP.GL01126 Results in Spermatid Mislocalization During Drosophila Spermatogenesis.
The Role of m6A Epigenetic Modification in the Treatment of Colorectal Cancer Immune Checkpoint Inhibitors.
Insufficient pyruvate in culture medium arrests mouse embryos at the first cleavage stage associated with abnormal epigenetic modifications.
Stimulation of Let-7 Maturation by Metformin Improved the Response to Tyrosine Kinase Inhibitor Therapy in an m6A Dependent Manner.
Critical role for cap-independent c-myc translation in progression of multiple myeloma.
Oxidative Stress Aggravates Apoptosis of Nucleus Pulposus Cells through m6A Modification of MAT2A Pre-mRNA by METTL16.
Construction and investigation of β3GNT2-associated regulatory network in esophageal carcinoma.
LncRNA LINC00942 promotes chemoresistance in gastric cancer by suppressing MSI2 degradation to enhance c-Myc mRNA stability.
Identification of Molecular Subtypes and Potential Small-Molecule Drugs for Esophagus Cancer Treatment Based on m6A Regulators.

J Exp Clin Cancer Res. 2022 Jan 25. 41(1): 36

The essential roles of m6A RNA modification to stimulate ENO1-dependent glycolysis and tumorigenesis in lung adenocarcinoma.

Lifang Ma, Xiangfei Xue, Xiao Zhang, Keke Yu, Xin Xu, Xiaoting Tian, Yayou Miao, Fanyu Meng, Xiaoxin Liu, Susu Guo, Shiyu Qiu, Yikun Wang, Jiangtao Cui, Wanxin Guo, You Li, Jinjing Xia, Yongchun Yu, Jiayi Wang.

   BACKGROUND: Lung adenocarcinoma (LUAD)  is the most common subtype of lung cancer. Patient prognosis is poor, and the existing therapeutic strategies for LUAD are far from satisfactory. Recently, targeting N6-methyladenosine (m6A) modification of RNA has been suggested as a potential strategy to impede tumor progression. However, the roles of m6A modification in LUAD tumorigenesis is unknown.
METHODS: Global m6A levels and expressions of m6A writers, erasers and readers were evaluated by RNA methylation assay, dot blot, immunoblotting, immunohistochemistry and ELISA in human LUAD, mouse models and cell lines. Cell viability, 3D-spheroid generation, in vivo LUAD formation, experiments in cell- and patient-derived xenograft mice and survival analysis were conducted to explore the impact of m6A on LUAD. The RNA-protein interactions, translation, putative m6A sites and glycolysis were explored in the investigation of the mechanism underlying how m6A stimulates tumorigenesis.
RESULTS: The elevation of global m6A level in most human LUAD specimens resulted from the combined upregulation of m6A writer methyltransferase 3 (METTL3) and downregulation of eraser alkB homolog 5 (ALKBH5). Elevated global m6A level was associated with a poor overall survival in LUAD patients. Reducing m6A levels by knocking out METTL3 and overexpressing ALKBH5 suppressed 3D-spheroid generation in LUAD cells and intra-pulmonary tumor formation in mice. Mechanistically, m6A-dependent stimulation of glycolysis and tumorigenesis occurred via enolase 1 (ENO1). ENO1 mRNA was m6A methylated at 359 A, which facilitated it's binding with the m6A reader YTH N6-methyladenosine RNA binding protein 1 (YTHDF1) and resulted in enhanced translation of ENO1. ENO1 positively correlated with METTL3 and global m6A levels, and negatively correlated with ALKBH5 in human LUAD. In addition, m6A-dependent elevation of ENO1 was associated with LUAD progression. In preclinical models, tumors with a higher global m6A level showed a more sensitive response to the inhibition of pan-methylation, glycolysis and ENO activity in LUAD.
CONCLUSIONS: The m6A-dependent stimulation of glycolysis and tumorigenesis in LUAD is at least partially orchestrated by the upregulation of METTL3, downregulation of ALKBH5, and stimulation of YTHDF1-mediated ENO1 translation. Blocking this mechanism may represent a potential treatment strategy for m6A-dependent LUAD.

Keywords:  ALKBH5; METTL3; RNA-protein interaction; YTHDF1; lung cancer; translation

DOI:  https://doi.org/10.1186/s13046-021-02200-5
RNA Biol. 2022 Jan;19(1): 132-142

The multifaceted functions of the Fat mass and Obesity-associated protein (FTO) in normal and cancer cells.

Sébastien Relier, Eric Rivals, Alexandre David.

  The last decade has seen mRNA modification emerge as a new layer of gene expression regulation. The Fat mass and obesity-associated protein (FTO) was the first identified eraser of N6-methyladenosine (m6A) adducts, the most widespread modification in eukaryotic messenger RNA. This discovery, of a reversible and dynamic RNA modification, aided by recent technological advances in RNA mass spectrometry and sequencing has led to the birth of the field of epitranscriptomics. FTO crystallized much of the attention of epitranscriptomics researchers and resulted in the publication of numerous, yet contradictory, studies describing the regulatory role of FTO in gene expression and central biological processes. These incongruities may be explained by a wide spectrum of FTO substrates and RNA sequence preferences: FTO binds multiple RNA species (mRNA, snRNA and tRNA) and can demethylate internal m6A in mRNA and snRNA, N6,2'-O-dimethyladenosine (m6Am) adjacent to the mRNA cap, and N1-methyladenosine (m1A) in tRNA. Here, we review current knowledge related to FTO function in healthy and cancer cells. In particular, we emphasize the divergent role(s) attributed to FTO in different tissues and subcellular and molecular contexts.

Keywords:  FTO; N6-methyladenosine; RNA modification; cancer; epitranscriptomics; transcription

DOI:  https://doi.org/10.1080/15476286.2021.2016203
Front Genet. 2021 ;12 825109

Current Advances in N6-Methyladenosine Methylation Modification During Bladder Cancer.

Qiang Liu.

  N6-methyladenosine (m6A) is a dynamic, reversible post-transcriptional modification, and the most common internal modification of eukaryotic messenger RNA (mRNA). Considerable evidence now shows that m6A alters gene expression, thereby regulating cell self-renewal, differentiation, invasion, and apoptotic processes. M6A methylation disorders are directly related to abnormal RNA metabolism, which may lead to tumor formation. M6A methyltransferase is the dominant catalyst during m6A modification; it removes m6A demethylase, promotes recognition by m6A binding proteins, and regulates mRNA metabolic processes. Bladder cancer (BC) is a urinary system malignant tumor, with complex etiology and high incidence rates. A well-differentiated or moderately differentiated pathological type at initial diagnosis accounts for most patients with BC. For differentiated superficial bladder urothelial carcinoma, the prognosis is normally good after surgery. However, due to poor epithelial cell differentiation, BC urothelial cell proliferation and infiltration may lead to invasive or metastatic BC, which lowers the 5-years survival rate and significantly affects clinical treatments in elderly patients. Here, we review the latest progress in m6A RNA methylation research and investigate its regulation on BC occurrence and development.

Keywords:  N6-methyladenosine; bladder cancer; methylation; “erasers”; “readers”; “writers”

DOI:  https://doi.org/10.3389/fgene.2021.825109
Front Cell Dev Biol. 2021 ;9 794801

Characterization of m6A Regulator-Mediated Methylation Modification Patterns and Tumor Microenvironment Infiltration in Ovarian Cancer.

Yihong Luo, Xiang Sun, Jian Xiong.

  Introduction: Studies have demonstrated the epigenetic regulation of immune responses in various cancers. However, little is known about the RNA N6-methyladenosine (m6A) modification patterns of the microenvironment (TME) cell infiltration in ovarian cancer (OC). Methods: We evaluated the correlation between m6A modification patterns and TME cell infiltration based on 459 OC samples from the Cancer Genome Atlas and Gene-Expression Omnibus database. We constructed an m6Ascore system to quantify m6A modification patterns using principal component analysis. Results: Based on unsupervised clustering, three m6A modification patterns were identified. Gene set variation analysis showed that the antigen presentation signal pathway, the NOTCH signaling pathway, and the metabolism-related pathway differed significantly across m6A modificaiton patterns. The m6Ascore is closely correlated with TME cell infiltration. OC patients with lower m6Ascores had worse outcomes. There was better risk stratification with combined m6Ascore and tumor mutation burden. The responses to immune checkpoint inhibitor treatment significantly differed between high and low m6Ascore groups. Conclusion: M6A modification plays an essential role in TME cell infiltration in OC. Evaluating the m6A modification patterns in OC patients could enhance our understanding of TME infiltration characterization and guide immunotherapy strategies.

Keywords:  N6-methyladenosine; RNA methylation; immunotherapy; microenvironment; ovairan cancer

DOI:  https://doi.org/10.3389/fcell.2021.794801
J Oncol. 2022 ;2022 3472745

β-Elemene Restrains PTEN mRNA Degradation to Restrain the Growth of Lung Cancer Cells via METTL3-Mediated N6 Methyladenosine Modification.

Yuxu Feng, Chenchen Li, Siwen Liu, Fei Yan, Yue Teng, Xiaoyou Li, Yan Sun.

Lung cancer is one of the most fatal malignancies and the leading cause of cancer death worldwide. β-Elemene, a well-known anticancer drug, has drawn a great deal of attention from researchers attributed to its limited side impacts. N6-Methyladenosine (m6A) modification is the most common RNA modification and plays a vital role in the pathogenesis of multiple tumors. However, the functional link between β-elemene and the m6A modification in lung cancer development remains unexplored. In this study, we investigated whether m6A modification was responsible for the impacts of β-elemene on lung cancer. Firstly, outcomes suggested that β-elemene restrained the malignant behaviors of A549 together with H1299 cells. Thereafter, we observed that β-elemene markedly regulated METTL3, YTHDF1, and YTHDC1 among various m6A modulators. METTL3 was selected for further study because of its oncogenic function in lung cancer. RT-qRCR and western blot assays exhibited that the mRNA and protein expression levels of METTL3 were lessened by the administration of β-elemene. Mechanistically, β-elemene exerted the restrictive impacts on the cell growth of lung cancer in vivo and in vitro through targeting METTL3. More importantly, β-elemene contributed to the augmented PTEN expression via suppressing its m6A modification. To sum up, we provided strong clues that β-elemene promoted PTEN expression to retard lung cancer progression by the regulation of METTL3-mediated m6A modification.

DOI: https://doi.org/10.1155/2022/3472745
Oncogene. 2022 Jan 24.

YTHDF2 promotes multiple myeloma cell proliferation via STAT5A/MAP2K2/p-ERK axis.

Zhen Hua, Rongfang Wei, Mengjie Guo, Zigen Lin, Xichao Yu, Xinying Li, Chunyan Gu, Ye Yang.

Multiple myeloma (MM) is still incurable partially due to lacking effective therapeutic targets. Aberrant N6-methyladenosine (m6A) RNA modification plays a vital role in many cancers, however few researches are executed in MM. We first screened the m6A-related genes in MM patient cohorts and correlated these genes with patient outcomes. We found that YTHDF2, a well-recognized m6A reader, was increased in MM patients and associated with poor outcomes. Decreased YTHDF2 expression hampered MM cell proliferation in vitro and in vivo, while enforced YTHDF2 expression reversed those effects. The analyses of m6A-RIP-seq and RIP-PCR indicated that STAT5A was the downstream target of YTHDF2, which was binding to the m6A modification site of STAT5A to promote its mRNA degradation. ChIP-seq and PCR assays revealed that STAT5A suppressed MM cell proliferation by occupying the transcription site of MAP2K2 to decrease ERK phosphorylation. In addition, we confirmed that YTHDF2 mediated the unphosphorylated form of STAT5A to inhibit the expression of MAP2K2/p-ERK. In conclusion, our study highlights that YTHDF2/STAT5A/MAP2K2/p-ERK axis plays a key role in MM proliferation and targeting YTHDF2 may be a promising therapeutic strategy.

DOI: https://doi.org/10.1038/s41388-022-02191-3
Bioengineered. 2022 Feb;13(2): 3093-3107

N6-methyladenosine-induced SVIL antisense RNA 1 restrains lung adenocarcinoma cell proliferation by destabilizing E2F1.

Zedong Hu, Liang Zhu, Yilin Zhang, Bing Chen.

  Accumulating evidence indicates that N6-methyladenosine (m6A) and long noncoding RNAs (lncRNAs) play crucial roles in cancer development. However, the biological roles of m6A and lncRNAs in lung cancer tumorigenesis are largely unknown. In this study, SVIL antisense RNA 1 (SVIL-AS1) was downregulated in lung adenocarcinoma (LUAD) tissues and was associated with a favorable prognosis in patients with LUAD. SVIL-AS1 overexpression suppressed LUAD cell proliferation and blocked cell cycle arrest. Mechanistically, METTL3 increased the m6A modification and transcript stability of SVIL-AS1. The enhanced SVIL-AS1 expression mediated by METTL3 suppressed E2F1 and E2F1-target genes. Moreover, SVIL-AS1 accelerated E2F1 degradation. The reduction in cell proliferation induced by SVIL-AS1 overexpression could be rescued by E2F1 overexpression or METTL3 knockdown. In conclusion, our work demonstrated the role and mechanism of METTL3-induced SVIL-AS1 in LUAD, which connects m6A and lncRNA in lung cancer carcinogenesis.

Keywords:  E2F1; METTL3; SVIL-AS1; degradation; lung adenocarcinoma; m6A

DOI:  https://doi.org/10.1080/21655979.2022.2025697
Front Immunol. 2021 ;12 762243

m6A Regulators Mediated Methylation Modification Patterns and Tumor Microenvironment Infiltration Characterization In Nasopharyngeal Carcinoma.

Zijian Liu, Jinlan He, Jiaqi Han, Jiangping Yang, Wenjun Liao, Nianyong Chen.

   Background: The role of RNA N6-methyladenosine (m6A) modification in tumor progression and metastasis has been demonstrated. Nonetheless, potential biological function of m6A modification patterns in nasopharyngeal carcinoma (NPC) remains unknown.
Methods: The m6A modification patterns were comprehensively evaluated based on 26 m6A regulators in NPC, and m6A subtype and also m6A score were identified and systematically correlated with representative tumor characteristics.
Results: Two distinct m6A subtypes were determined and were highly consistent with immune activated and immune suppressed phenotypes, respectively. More representative m6A scores of individual tumors could predict tumor microenvironment (TME) infiltration, mRNA based stemness index (mRNAsi), EBV gene expression, genetic variation, and prognosis of NPC patients. Low m6A score, characterized by activation of immunity and suppression of mRNAsi and EBV gene, indicated an activated TME phenotype and better PFS and also lower risk of recurrence and metastasis. High m6A score, characterized by activation of Wnt and NF-κB signaling pathway and lack of effective immune infiltration, indicated an immune suppressed TME phenotype and poorer survival. Low m6A score was also correlated with increased tumor mutation burden (TMB) and better response to immunotherapy, and vice versa. A significant therapeutic advantage in patients with low m6A score was confirmed with an anti-PDL1 immunotherapy cohort.
Conclusions: m6A patterns played an important role in the diversity and complexity of TME. m6A score could be used to evaluate the m6A pattern of individual tumor to enhance our understanding of TME infiltration and guide more effective immunotherapy strategies.

Keywords:  TIDE; immunotherapy; m6A score; molecular subtype; nasopharyngeal carcinoma; tumor microenvironment

DOI:  https://doi.org/10.3389/fimmu.2021.762243
Mol Cell. 2022 Jan 17. pii: S1097-2765(21)01143-6. [Epub ahead of print]

scDART-seq reveals distinct m6A signatures and mRNA methylation heterogeneity in single cells.

Matthew Tegowski, Mathieu N Flamand, Kate D Meyer.

N6-methyladenosine (m6A) is an abundant RNA modification that plays critical roles in RNA regulation and cellular function. Global m6A profiling has revealed important aspects of m6A distribution and function, but to date such studies have been restricted to large populations of cells. Here, we develop a method to identify m6A sites transcriptome-wide in single cells. We uncover surprising heterogeneity in the presence and abundance of m6A sites across individual cells and identify differentially methylated mRNAs across the cell cycle. Additionally, we show that cellular subpopulations can be distinguished based on their RNA methylation signatures, independent from gene expression. These studies reveal fundamental features of m6A that have been missed by m6A profiling of bulk cells and suggest the presence of cell-intrinsic mechanisms for m6A deposition.

DOI: https://doi.org/10.1016/j.molcel.2021.12.038
Front Cell Dev Biol. 2021 ;9 760912

Comprehensive Analysis of Differentially Expressed Profiles of mRNA N6-Methyladenosine in Colorectal Cancer.

Na Li, Qin Guo, Qiao Zhang, Bai-Jun Chen, Xiao-An Li, Yan Zhou.

  Aim: To comprehensively profile the landscape of the mRNA N6-methyladenosine (m6A) modification in human colorectal cancer (CRC). Methods: Methylated RNA immunoprecipitation sequencing (MeRIP-seq) was explored to compare the difference in mRNA N6-methyladenosine (m6A) methylation between CRC tissues and adjacent normal control (NC) tissue. RNA-sequencing (RNA-seq) was performed to transcribe differentially expressed mRNAs. Conjoint analysis of MeRIP-seq and RNA-seq data was conducted to predict RNA-binding proteins (RBPs). Results: MeRIP-seq identified 1110 differentially m6A methylated sites (DMMSs) and 980 differentially m6A methylated genes (DMMGs) in CRC, with 50.13% of all modified genes showing unique m6A-modified peaks in CRC. RNA-seq showed 915 upregulated genes and 1463 downregulated genes in CRC. QRT-PCR verified the RNA-seq results by detecting the expression of some mRNAs. Conjoint analysis of MeRIP-seq and RNA-seq identified 400 differentially m6A methylated and expressed genes (DEGs), and pathway analysis detected that DMMGs and DEGs were closely related to cancer. After analyzing these DMMGs and DEGs through the GEPIA database, we found that the expression of B3GNT6, DKC1, SRPK1, and RIMKLB were associated with prognosis, and the expression of B3GNT6 and RIMKLB were associated with clinical stage. 17 RBPs were identified based on the DMMGs and DEGs, among which FXR1, FXR2, FMR1, IGF2BP2, IGF2BP3, and SRSF1 were obviously highly expressed in CRC, and FMR1, IGF2BP2, and IGF2BP3 were closely related to methylation, and might be involved in the development of CRC. Conclusion: This study comprehensively profiled m6A modification of mRNAs in CRC, which revealed possible mechanisms of m6A-mediated gene expression regulation.

Keywords:  MeRIP sequencing; RNA-binding protein; RNA-seq; colorectal cancer; m6A

DOI:  https://doi.org/10.3389/fcell.2021.760912
Sci Rep. 2022 Jan 24. 12(1): 1222

Prognostic implications of N6-methyladenosine RNA regulators in breast cancer.

Jiaojiao Tai, Linbang Wang, Hao Guo, Ziqiang Yan, Jingkun Liu.

The significance of N6-methyladenosine (m6A) RNA modifications in the progression of breast cancer (BC) has been recognised. However, their potential role and mechanism of action in the tumour microenvironment (TME) and immune response has not been demonstrated. Thus, the role of m6A regulators and their downstream target gene components in BC remain to be explored. In this study, we used a series of bioinformatics methods and experiments to conduct exploratory research on the possible role of m6A regulators in BC. First, two regulatory modes of immune activation and inactivation were determined by tumour classification. The TME, immune cell infiltration, and gene set variation analysis results confirmed the reliability of this pattern. The prognostic model of the m6A regulator was established by the least absolute shrinkage and selection operator and univariate and multivariate Cox analyses, with the two regulators most closely related to survival verified by real-time quantitative reverse transcription polymerase chain reaction. Next, the prognostic m6A regulator identified in the model was crossed with the differential copy number of variant genes in invasive BC (IBC), and it was determined that YTHDF1 was a hub regulator. Subsequently, single-cell analysis revealed the expression patterns of m6A regulators in different IBC cell populations and found that YTHDF1 had significantly higher expression in immune-related IBC cells. Therefore, we selected the intersection of the BC differential expression gene set and the differential expression gene set of a cell line with knocked-down YTHDF1 in literature to identify downstream target genes of YTHDF1, in which we found IFI6, EIR, and SPTBN1. A polymerase chain reaction was conducted to verify the results. Finally, we confirmed the role of YTHDF1 as a potential prognostic biomarker through pan-cancer analysis. Furthermore, our findings revealed that YTHDF1 can serve as a new molecular marker for BC immunotherapy.

DOI: https://doi.org/10.1038/s41598-022-05125-x
Front Oncol. 2021 ;11 784127

The Interaction Between N6-Methyladenosine Modification and Non-Coding RNAs in Gastrointestinal Tract Cancers.

Lin Yao, Chang-Feng Man, Rong He, Lian He, Jia-Bin Huang, Shou-Yan Xiang, Zhe Dai, Xiao-Yan Wang, Yu Fan.

  N6-methyladenosine (m6A) is the most common epigenetic modification of eukaryotic RNA, which can participate in the growth and development of the body and a variety of physiological and disease processes by affecting the splicing, processing, localization, transport, translation, and degradation of RNA. Increasing evidence shows that non-coding RNAs, particularly microRNA, long non-coding RNA, and circular RNA, can also regulate the RNA m6A modification process by affecting the expression of m6A-related enzymes. The interaction between m6A modification and non-coding RNAs provides a new perspective for the exploration of the potential mechanism of tumor genesis and development. In this review, we summarize the potential mechanisms and effects of m6A and non-coding RNAs in gastrointestinal tract cancers.

Keywords:  colorectal cancer; gastrointestinal tract cancers; long non-coding RNA (lncRNA); m6A modification; non-coding RNAs (ncRNAs)

DOI:  https://doi.org/10.3389/fonc.2021.784127
Mol Ther Nucleic Acids. 2022 Mar 08. 27 598-610

Comprehensive analysis of N6-methylandenosine regulators and m6A-related RNAs as prognosis factors in colorectal cancer.

Wei Li, Yingchao Gao, Xiaojing Jin, Haobo Wang, Tianhao Lan, Ming Wei, Weitao Yan, Guiqi Wang, Zhongxin Li, Zengren Zhao, Xia Jiang.

  Colorectal cancer (CRC) is one of the most common malignancies and has been a leading cause of cancer-related death worldwide in recent years. N6-methyladenosine (m6A) methylation is the most abundant epigenetic modification of various types of RNAs, and it plays a vital role in promoting cancer development. Here, we obtained SNV and transcriptome data of CRC from The Cancer Genome Atlas (TCGA). We demonstrated that most m6A methylation regulators were aberrantly expressed in individuals with CRC. The abnormal expression of m6A regulators was caused by their different copy number variation (CNV) patterns, and alteration of m6A regulators was significantly correlated with prognosis and tumor stage. By using weighted coexpression network analysis (WGCNA), we identified m6A-related long noncoding RNAs (lncRNAs) and mRNAs; then we used least absolute shrinkage and selection operator (LASSO) Cox regression analysis to construct m6A-related lncRNA and mRNA prognostic signatures in the TCGA dataset. Furthermore, a nomogram with clinicopathological features, lncRNA risk scores, and mRNA risk scores was established, which showed a strong ability to forecast the overall survival of the individuals with CRC in training and testing sets. In conclusion, m6A methylation regulators played a vital role in affecting the prognosis of subjects with CRC, and m6A-related lncRNAs and mRNAs revealed underlying mechanisms in CRC tumorigenesis and progression.

Keywords:  N6-methylandenosine; TCGA; WGCNA; colorectal cancer; m6A-related lncRNA; m6A-related mRNA; nomogram; prognosis signature

DOI:  https://doi.org/10.1016/j.omtn.2021.12.007
Cardiovasc Drugs Ther. 2022 Jan 23.

Mechanism of METTL3-Mediated m6A Modification in Cardiomyocyte Pyroptosis and Myocardial Ischemia-Reperfusion Injury.

Xiang Wang, Yi Li, Jiahan Li, Shiguo Li, Fang Wang.

   OBJECTIVE: Myocardial ischemia/reperfusion (MI/R) injury is a complicated pathophysiological process associated with cardiomyocyte pyroptosis. Methyltransferase-like protein 3 (METTL3) catalyzes the formation of N6-methyl-adenosine (m6A) and participates in various biological processes. This study probed into the mechanism of METTL3 in cardiomyocyte pyroptosis in MI/R injury.
METHODS: A rat model of MI/R was established. Rat cardiomyocytes were subjected to oxygen-glucose deprivation/reoxygenation (OGD/R) treatment for the establishment of a cell model in vitro. METTL3 expression in myocardial tissues of MI/R rats and OGD/R-treated cardiomyocytes was determined using RT-qPCR and Western blot. The pathological changes of rat myocardial tissues were observed using hematoxylin and eosin staining. The positive expression of NLRP3 in myocardial tissues or cardiomyocytes was observed through immunohistochemistry or immunofluorescence. The activity of caspase-1 was measured using the colorimetric method. The expressions of GSDMD and cleaved caspase-1, as well as the levels of IL-1β and IL-18 in rat myocardial tissues or cardiomyocytes were determined. m6A modification level was quantified. The binding relationship between pri-miR-143-3p and DGCR8 and the enrichment of m6A on pri-miR-143-3p were detected. The binding relationship between miR-143-3p and protein kinase C epsilon (PRKCE) was verified.
RESULTS: METTL3 expression was elevated in MI/R rats and OGD/R cardiomyocytes. METTL3 silencing alleviated myocardial injury, reduced the number of NLRP3-positive cardiomyocytes, suppressed caspase-1 activity, decreased the protein levels of GSDMD-N and cleaved caspase-1, and decreased IL-1β and IL-18 levels. METTL3 increased the total m6A level in MI/R rats and injured cardiomyocytes, promoted DGCR8 binding to pri-miR-143-3p, and enhanced miR-143-3p expression. miR-143-3p suppressed PRKCE transcription, and miR-143-3p overexpression reversed the inhibitory effect of METTL3 silencing on cardiomyocyte pyroptosis.
CONCLUSION: METTL3 promoted DGCR8 binding to pri-miR-143-3p through m6A modification, thus enhancing miR-143-3p expression to inhibit PRKCE transcription and further aggravating cardiomyocyte pyroptosis and MI/R injury.

Keywords:  METTL3; Pri-miR-143-3p; Pyroptosisdainty myocardial ischemia/reperfusion; m6A modification; miR-143-3p

DOI:  https://doi.org/10.1007/s10557-021-07300-0
MicroPubl Biol. 2022 ;2022

Ubiquitous Knockdown of Mettl3 using TRiP.GL01126 Results in Spermatid Mislocalization During Drosophila Spermatogenesis.

Lauren Rowe, Antonio L Rockwell.

METTL3, the enzyme that catalyzes the m6A RNA modification in Drosophila is highly conserved and essential in various eukaryotic organisms. Mettl3 and its homologs have been linked to biological processes such as gametogenesis. We focused on characterizing the role of METTL3 in Drosophila spermatogenesis. We used the Gal4-UAS system to ubiquitously knockdown Mettl3 in both somatic cyst cells and germline cells. Using immunostaining and confocal microscopy, we found spermatid bundles mislocalize in testes that contain the morphologically abnormal swollen apical tip. Our result suggests Mettl3 knockdown using TRiP.GL01126 results in spermatogenesis aberrations.

DOI: https://doi.org/10.17912/micropub.biology.000511
Front Immunol. 2021 ;12 802049

The Role of m6A Epigenetic Modification in the Treatment of Colorectal Cancer Immune Checkpoint Inhibitors.

Huan Tong, He Wei, Alhaji Osman Smith, Juan Huang.

  Tumor immunotherapy, one of the efficient therapies in cancers, has been called to the scientific community's increasing attention lately. Among them, immune checkpoint inhibitors, providing entirely new modalities to treat cancer by leveraging the patient's immune system. They are first-line treatments for varieties of advanced malignancy, such as melanoma, gastrointestinal tumor, esophageal cancer. Although immune checkpoint inhibitors (ICIs) treatment has been successful in different cancers, drug resistance and relapses are common, such as in colorectal cancer. Therefore, it is necessary to improve the efficacy of immune checkpoint therapy for cancer patients who do not respond or lowly response to current treatments. N6-methyladenosine (m6A), as a critical regulator of transcript expression, is the most frequently internal modification of mRNA in the human body. Recently, it has been proposed that m6A epigenetic modification is a potential driver of tumor drug resistance. In this report, we will briefly outline the relevant mechanisms, general treatment status of immune checkpoint inhibitors in colorectal cancer, how m6A epigenetic modifications regulate the response of ICIs in CRC and provide new strategies for overcoming the resistance of ICIs in CRC.

Keywords:  ICB; colorectal cancer; m6A epigenetics; overcome resistance; resistance

DOI:  https://doi.org/10.3389/fimmu.2021.802049
Theriogenology. 2022 Jan 18. pii: S0093-691X(22)00015-2. [Epub ahead of print]181 119-125

Insufficient pyruvate in culture medium arrests mouse embryos at the first cleavage stage associated with abnormal epigenetic modifications.

Pan Li, Hengye Zhang, Ke Yan, Lumin Sui, Ya Du, Jiahao Hu, Huiyan Xu, Xiaogan Yang, Xingwei Liang.

  Energy is essential for early embryogenesis, and fertilized eggs can successfully develop to blastocyst in in vitro culture medium with an appropriate energy supply. Conversely, embryonic development is negatively affected by a suboptimal energy supply. We previously observed that a low level of pyruvate greatly arrests mouse embryos at the 2-cell stage. However, how methylation modifications are affected at this specific stage remains unknown. In this study, we found that mouse embryos could timely develop to the 4-cell stage in K+simplex optimized medium (KSOM) with control level of pyruvate, but embryos were significantly arrested at the 2-cell stage when pyruvate was reduced to 0.2-fold of the control level. Moreover, the fluorescence intensities of 5 mC, H3K4me2, H3K9me2 and H3K27me2 in the 2-cell stage embryos of the 0.2-fold pyruvate group were notedly lower than those of the control group, but N6-methyladenosine (m6A) fluorescence intensity was higher, suggesting that global genomic DNA, histone and m6A methylation modifications are disrupted with low levels of pyruvate. Consistently, the mRNA levels of genes related to DNA methylation, histone methylation and m6A modifications were also disturbed in the 2-cell stage embryos cultured with low levels of pyruvate. In summary, our findings demonstrate that insufficient pyruvate in culture medium results in mouse embryonic developmental arrest, at least in part due to defects in methylation modifications.

Keywords:  Developmental arrest; Early embryogenesis; Epigenetic modification; In vitro culture; Methylation; Pyruvate

DOI:  https://doi.org/10.1016/j.theriogenology.2022.01.015
Front Oncol. 2021 ;11 731561

Stimulation of Let-7 Maturation by Metformin Improved the Response to Tyrosine Kinase Inhibitor Therapy in an m6A Dependent Manner.

Kai Li, Shan Gao, Lei Ma, Ye Sun, Zi-Yang Peng, Jie Wu, Ning Du, Hong Ren, Shou-Ching Tang, Xin Sun.

  The molecular mechanism of the tyrosine kinase inhibitor (TKI) resistant lung adenocarcinoma is currently unclear, and the role of methylated adenosine at the N6 position in the resistance of cancer stem cells (CSCs) therapy is unknown. This study identified a novel and effective strategy to enhance TKIs therapy response. We first confirmed the sensitization of Metformin enforcing on Osimertinib treatment and revealed the mature miRNAs signatures of the Osimertinib resistant H1975 and HCC827 cells. Let-7b expression was stimulated when adding Metformin and then increasing the therapy sensitivity by decreasing the stem cell groups expanding. Methyltransferase-like 3 (METTL3) increased the pri-Let-7b, decreased both the pre-Let-7b and mature Let-7b, attenuating the Let-7b controlling of stem cell renewal. The addition of Metformin increased the bindings of DNA methyltransferase-3a/b (DNMT3a/b) to the METTL3 promoter. With the help of the readers of NKAP and HNRNPA2B1, the cluster mediated m6A formation on pri-Let-7b processing increased the mature Let-7b, the key player in suppressing Notch signaling and re-captivating Osimertinib treatment. We revealed that the maturation processing signaling stimulated the methylation regulation of the miRNAs, and may determine the stemness control of the therapy resistance. Our findings may open up future drug development, targeting this pathway for lung cancer patients.

Keywords:  cancer stem-like cells; miRNAs maturation; n6-methyladenosine; therapy resistance; tyrosine kinase inhibitor

DOI:  https://doi.org/10.3389/fonc.2021.731561
Mol Cancer Ther. 2022 Jan 27. pii: molcanther.0016.2021. [Epub ahead of print]

Critical role for cap-independent c-myc translation in progression of multiple myeloma.

Yijiang Shi, Fumou Sun, Yan Cheng, Brent Holmes, Binod Dhakal, Joseph F Gera, Siegfried Janz, Alan Lichtenstein.

Dysregulated c-myc is a determinant of multiple myeloma (MM) progression. Translation of c-myc can be achieved by an mTOR-mediated, cap-dependent mechanism or a cap-independent mechanism where a sequence in the 5'UTR of mRNA, termed the IRES (internal ribosome entry site), recruits the 40S ribosomal subunit. This mechanism requires the RNA-binding factor hnRNP A1 (A1) and becomes critical when cap-dependent translation is inhibited during endoplasmic reticulum (ER) stress. We, thus, studied the role of A1 and the myc IRES in myeloma biology. A1 expression correlated with enhanced c-myc expression in patient samples. Expression of A1 in MM lines was mediated by c-myc itself, suggesting a positive feed-back circuit where myc induces A1 and A1 enhances myc translation. We then deleted the A1 gene in a myc-driven murine myeloma model. A1-deleted MM cells demonstrated down-regulated myc expression and were inhibited in their growth in vivo. Decreased myc expression was due to reduced translational efficiency and depressed IRES activity. We also studied the J007 inhibitor which prevents A1's interaction with the myc IRES. J007 inhibited myc translation and IRES activity and diminished myc expression in murine and human MM lines as well as primary samples. J007 also inhibited tumor outgrowth in mice after subcutaneous or intravenous challenge and prevented osteolytic bone disease. When c-myc was ectopically re-expressed in A1-deleted MM cells, tumor growth was re-established. These results support the critical role of A1-dependent myc IRES-translation in myeloma.

DOI: https://doi.org/10.1158/1535-7163.MCT-21-0016
Oxid Med Cell Longev. 2022 ;2022 4036274

Oxidative Stress Aggravates Apoptosis of Nucleus Pulposus Cells through m6A Modification of MAT2A Pre-mRNA by METTL16.

Peng-Bo Chen, Gui-Xun Shi, Tao Liu, Bo Li, Sheng-Dan Jiang, Xin-Feng Zheng, Lei-Sheng Jiang.

The process of intervertebral disc degeneration (IVDD) is complex, and its mechanism is considered multifactorial. Apoptosis of oxidative stressed nucleus pulposus cells (NPCs) should be a fundamental element in the pathogenesis of IVDD. In our pilot study, we found that the expression of MAT2A decreased, and METTL16 increased in the degenerative nucleus pulposus tissues. Previous studies have shown that the balance of splicing, maturation, and degradation of MAT2A pre-mRNA is regulated by METTL16 m6A modification. In the current study, we aimed to figure out whether this mechanism was involved in the aberrant apoptosis of NPCs and IVDD. Human NPCs were isolated and cultured under oxidative stress. An IVDD animal model was established. It showed that significantly higher METTL16 expression and lower MAT2A expression were seen in either the NPCs under oxidative stress or the degenerative discs of the animal model. MAT2A was inhibited with siRNA in vitro or cycloleucine in vivo. METTL16 was overexpressed with lentivirus in vitro or in vivo. Downregulation of MAT2A or upregulation of METTL16 aggravated nucleus pulposus cell apoptosis and disc disorganization. The balance of splicing, maturation, and degradation of MAT2A pre-mRNA was significantly inclined to degradation in the NPCs with the overexpression of METTL16. Increased apoptosis of NPCs under oxidative stress could be rescued by reducing the expression of METTL16 using siRNA with more maturation of MAT2A pre-mRNA. Collectively, oxidative stress aggravates apoptosis of NPCs through disrupting the balance of splicing, maturation, and degradation of MAT2A pre-mRNA, which is m6A modified by METTL16.

DOI: https://doi.org/10.1155/2022/4036274
Cell Mol Biol Lett. 2022 Jan 24. 27(1): 8

Construction and investigation of β3GNT2-associated regulatory network in esophageal carcinoma.

Zhiguo Luo, Qing Hu, Yuanhui Tang, Yahui Leng, Tian Tian, Shuangyue Tian, Chengyang Huang, Ao Liu, Xinzhou Deng, Li Shen.

   BACKGROUND: Glycosyltransferases play a crucial role in various cancers. β1, 3-N-acetylglucosaminyltransferase 2, a polylactosamine synthase, is an important member of the glycosyltransferase family. However, the biological function and regulatory mechanism of β3GNT2 in esophageal carcinoma (ESCA) is still poorly understood.
METHODS: The Cancer Genome Atlas and Genotype-Tissue Expression databases were used for gene expression and prognosis analysis. Quantitative real-time PCR, Western blot, and immunohistochemistry were performed to detect the expression of β3GNT2 in ESCA cell lines and tissues. In vitro assays and xenograft tumor models were utilized to evaluate the impact of β3GNT2 on ESCA progression. The downstream effectors and upstream regulators of β3GNT2 were predicted by online software and verified by functional experiments.
RESULTS: We found that β3GNT2 was highly expressed in ESCA tissues and positively correlated with poor prognosis in ESCA patients. β3GNT2 expression was closely associated with the tumor size, TNM stage, and overall survival of ESCA patients. Functionally, β3GNT2 promoted ESCA cell growth, migration, and invasion in vitro, as well as tumorigenesis in vivo. Mechanistically, β3GNT2 knockdown decreased the expression of the polylactosamine on EGFR. Knockdown of β3GNT2 also inhibited the JAK/STAT signaling pathway. Meanwhile, the JAK/STAT inhibitor could partly reverse the biological effects caused by β3GNT2 overexpression. Moreover, β3GNT2 expression was positively regulated by CREB1 and negatively regulated by miR-133b. Both CREB1 and miR-133b was involved in the β3GNT2-mediated ESCA progression.
CONCLUSIONS: Our study, for the first time, reveals the importance of β3GNT2 in ESCA progression and offers a potential therapeutic target for ESCA.

Keywords:  Esophageal carcinoma; Glycosyltransferase; Progression; β3GNT2

DOI:  https://doi.org/10.1186/s11658-022-00306-y
Clin Transl Med. 2022 Jan;12(1): e703

LncRNA LINC00942 promotes chemoresistance in gastric cancer by suppressing MSI2 degradation to enhance c-Myc mRNA stability.

Yiran Zhu, Bingluo Zhou, Xinyang Hu, Shilong Ying, Qiyin Zhou, Wenxia Xu, Lifeng Feng, Tianlun Hou, Xian Wang, Liyuan Zhu, Hongchuan Jin.

   BACKGROUND: Chemoresistance to cisplatin (DDP) remains a major challenge in advanced gastric cancer (GC) treatment. Although accumulating evidence suggests an association between dysregulation of long non-coding RNAs (lncRNAs) and chemoresistance, the regulatory functions and complexities of lncRNAs in modulating DDP-based chemotherapy in GC remain under-investigated. This study was designed to explore the critical chemoresistance-related lncRNAs in GC and identify novel therapeutic targets for patients with chemoresistant GC.
METHODS: Chemoresistance-related lncRNAs were identified through microarray and verified through a quantitative real-time polymerase chain reaction (qRT-PCR). Proteins bound by lncRNAs were identified through a human proteome array and validated through RNA immunoprecipitation (RIP) and RNA pull-down assays. Co-immunoprecipitation and ubiquitination assays were performed to explore the molecular mechanisms of the Musashi2 (MSI2) post-modification. The effects of LINC00942 (LNC942) and MSI2 on DDP-based chemotherapy were investigated through MTS, apoptosis assays and xenograft tumour formation in vivo.
RESULTS: LNC942 was found to be up-regulated in chemoresistant GC cells, and its high expression was positively correlated with the poor prognosis of patients with GC. Functional studies indicated that LNC942 confers chemoresistance to GC cells by impairing apoptosis and inducing stemness. Mechanically, LNC942 up-regulated the MSI2 expression by preventing its interaction with SCFβ-TRCP E3 ubiquitin ligase, eventually inhibiting ubiquitination. Then, LNC942 stabilized c-Myc mRNA in an N6-methyladenosine (m6 A)-dependent manner. As a potential m6 A recognition protein, MSI2 stabilized c-Myc mRNA with m6 A modifications. Moreover, inhibition of the LNC942-MSI2-c-Myc axis was found to restore chemosensitivity both in vitro and in vivo.
CONCLUSIONS: These results uncover a chemoresistant accelerating function of LNC942 in GC, and disrupting the LNC942-MSI2-c-Myc axis could be a novel therapeutic strategy for GC patients undergoing chemoresistance.

Keywords:  LINC00942 (LNC942); Musashi2 (MSI2); c-Myc; chemoresistance; m6A

DOI:  https://doi.org/10.1002/ctm2.703
J Oncol. 2022 ;2022 5490461

Identification of Molecular Subtypes and Potential Small-Molecule Drugs for Esophagus Cancer Treatment Based on m6A Regulators.

Jianjun Li, Hongbo Zhu, Qiao Yang, Hua Xiao, Haibiao Wu, Zhe Fang, Wenjun Li, Manbo Cai.

Background: Esophagus cancer (ESCA) is the sixth most frequent cancer in males, with 5-year overall survival of 15%-25%. RNA modifications function critically in cancer progression, and m6A regulators are associated with ESCA prognosis. This study further revealed correlations between m6A and ESCA development.
Methods: Univariate Cox regression analysis and consensus clustering were applied to determine molecular subtypes. Functional pathways and gene ontology terms were enriched by gene set enrichment analysis. Protein-protein interaction (PPI) analysis on differentially expressed genes (DEGs) was conducted for hub gene screening. Public drug databases were employed to study the interactions between hub genes and small molecules.
Results: Three molecular subtypes related to ESCA prognosis were determined. Based on multiple analyses among molecular subtypes, 146 DEGs were screened, and a PPT network of 15 hub genes was visualized. Finally, 8 potential small-molecule drugs (BMS-754807, gefitinib, neratinib, zuclopenthixol, puromycin, sulfasalazine, and imatinib) were identified for treating ESCA.
Conclusions: This study applied a new approach to analyzing the relation between m6A and ESCA prognosis, providing a reference for exploring potential targets and drugs for ESCA treatment.

DOI: https://doi.org/10.1155/2022/5490461