bims-rimeca 2021-08-22 papers

bims-rimeca

Biomed News

on RNA methylation in cancer

Issue of 2021–08–22
twenty papers selected by
Sk Ramiz Islam, Saha Institute of Nuclear Physics

Aging-Associated Differences in Epitranscriptomic m6A Regulation in Response to Acute Cardiac Ischemia/Reperfusion Injury in Female Mice.
The germinal center reaction depends on RNA methylation and divergent functions of specific methyl readers.
METTL3-mediated m6A methylation of ASPM drives hepatocellular carcinoma cells growth and metastasis.
Lipopolysaccharide Alters the m6A Epitranscriptomic Tagging of RNAs in Cardiac Tissue.
N6-Methyladenosine Regulators and Related LncRNAs Are Potential to be Prognostic Markers for Uveal Melanoma and Indicators of Tumor Microenvironment Remodeling.
m6A-express: uncovering complex and condition-specific m6A regulation of gene expression.
IGF2BP2-modified circular RNA circARHGAP12 promotes cervical cancer progression by interacting m6A/FOXM1 manner.
Characteristic of molecular subtypes in lung adenocarcinoma based on m6A RNA methylation modification and immune microenvironment.
Identification of a robust signature for clinical outcomes and immunotherapy response in gastric cancer: based on N6-methyladenosine related long noncoding RNAs.
m6A Methyltransferase 3 Promotes the Proliferation and Migration of Gastric Cancer Cells through the m6A Modification of YAP1.
N6-methyladenosine binding induces a metal-centered rearrangement that activates the human RNA demethylase Alkbh5.
m6 A transferase KIAA1429-stabilized LINC00958 accelerates gastric cancer aerobic glycolysis through targeting GLUT1.
Quantitative analysis of m6A RNA modification by LC-MS.
Human umbilical cord mesenchymal stem cells deliver exogenous miR-26a-5p via exosomes to inhibit nucleus pulposus cell pyroptosis through METTL14/NLRP3.
YTHDF1 and YTHDF2 are associated with better patient survival and an inflamed tumor-immune microenvironment in non-small-cell lung cancer.
Network analysis of miRNA targeting m6A-related genes in patients with esophageal cancer.
A seven-m6A regulator-related CpG site-based prognostic signature for endometrial carcinoma.
How RNA modifications regulate the antiviral response.
m5C-Related Signatures for Predicting Prognosis in Cutaneous Melanoma with Machine Learning.
NAT10-Mediated N4-Acetylcytidine of RNA Contributes to Post-transcriptional Regulation of Mouse Oocyte Maturation in vitro.

Front Pharmacol. 2021 ;12 654316

Aging-Associated Differences in Epitranscriptomic m6A Regulation in Response to Acute Cardiac Ischemia/Reperfusion Injury in Female Mice.

Xuan Su, Yan Shen, Yue Jin, Il-Man Kim, Neal L Weintraub, Yaoliang Tang.

  Elderly patients are more susceptible to ischemic injury. N6-methyladenosine (m6A) modification is the most abundant reversible epitranscriptomic modification in mammalian RNA and plays a vital role in many biological processes. However, it is unclear whether age difference impacts m6A RNA methylation in hearts and their response to acute myocardial ischemia/reperfusion (I/R) injury. In this study, we measured the global level of m6A RNA methylation as well as the expression of m6A RNA "writers" (methylation enzymes) and "erasers" (demethylation enzymes) in the hearts of young and elderly female mice undergone sham surgery or acute MI/R injury. We found that m6A RNA level and associate modifier gene expression was similar in intact young and old female hearts. However, young hearts show a significant reduction in m6A RNA while elderly hearts showed only a slight reduction in m6A RNA in response to acute I/R injury. To explore the mechanism of differential level of m6A RNA modification, we use qRT-PCR and Western blotting to compare the mRNA and protein expression of major m6A-related "writers" (Mettl3, Mettl14, and WTAP) and 'erasers" (ALKBH5 and FTO). Mettl3 mRNA and protein expression were significantly reduced in both young and elderly hearts. However, the levels of FTO's mRNA and protein were only significantly reduced in ischemic elderly hearts, and age-related downregulation of FTO may offset the effect of reduced Mettl3 on reduced m6A RNA level in the hearts of aging mice hearts with acute I/R injury, indicating aging-related differences in epitranscriptomic m6A regulation in hearts in response to acute I/R injury. To further investigate specific I/R related targets of Mettl3, we overexpressed Mettl3 in cardiomyocyte line (HL1) using lentiviral vector, and the m6A enrichment of Bcl2, Bax and PTEN were quantified with m6A RIP-qPCR, we found that m6A modification of PTEN mRNA decreased after in vitro hypoxia/reperfusion injury (iH/R) while Mettl3 augments m6A levels of both Bax and PTEN after iH/R, indicating that Bax and PTEN are target genes of Mettl3 under iH/R stress.

Keywords:  FTO; M6A RNA methylation; METTL3; aging; epitranscriptomics; myocardial ischemia/reperfusion; pten

DOI:  https://doi.org/10.3389/fphar.2021.654316
J Exp Med. 2021 Oct 04. pii: e20210360. [Epub ahead of print]218(10):

The germinal center reaction depends on RNA methylation and divergent functions of specific methyl readers.

Amalie C Grenov, Lihee Moss, Sarit Edelheit, Ross Cordiner, Dominik Schmiedel, Adi Biram, Jacob H Hanna, Torben Heick Jensen, Schraga Schwartz, Ziv Shulman.

Long-lasting immunity depends on the generation of protective antibodies through the germinal center (GC) reaction. N6-methyladenosine (m6A) modification of mRNAs by METTL3 activity modulates transcript lifetime primarily through the function of m6A readers; however, the physiological role of this molecular machinery in the GC remains unknown. Here, we show that m6A modifications by METTL3 are required for GC maintenance through the differential functions of m6A readers. Mettl3-deficient GC B cells exhibited reduced cell-cycle progression and decreased expression of proliferation- and oxidative phosphorylation-related genes. The m6A binder, IGF2BP3, was required for stabilization of Myc mRNA and expression of its target genes, whereas the m6A reader, YTHDF2, indirectly regulated the expression of the oxidative phosphorylation gene program. Our findings demonstrate how two independent gene networks that support critical GC functions are modulated by m6A through distinct mRNA binders.

DOI: https://doi.org/10.1084/jem.20210360
J Clin Lab Anal. 2021 Aug 16. e23931

METTL3-mediated m6A methylation of ASPM drives hepatocellular carcinoma cells growth and metastasis.

An Wang, Xiaofeng Chen, Dongen Li, Liang Yang, Jianshuai Jiang.

   BACKGROUND: Abnormal spindle-like microcephaly (ASPM) has been proved to participate in tumor progression. However, the underlying mechanism of ASPM in liver hepatocellular carcinoma (LIHC) remains elusive.
METHODS: The mRNA and protein expression were determined using Western blot and qRT-PCR, and the capacities of cells proliferation, migration, and invasion were evaluated by CCK-8, colony formation, wound healing, and transwell. MeRIP was performed to validate the interaction between ASPM and methyltransferase-like 3 (METTL3).
RESULTS: Herein, we found that ASPM was significantly upregulated in LIHC, and the high expression of ASPM was associated with poor LIHC prognosis. Furthermore, ASPM knockdown could suppress LIHC cells proliferation, migration, and invasion, while ASPM overexpression exerted reverse effect. Mechanistically, we revealed that the N6-methyladenosine (m6A) modification of ASPM mRNA mediated by METTL3 promoted its expression in LIHC. More importantly, silencing METTL3 suppressed LIHC cells proliferation, migration, and invasion, which could be retained by ASPM overexpression.
CONCLUSION: Collectively, our findings suggested that METTL3/ASPM axis could serve as a novel promising therapeutic candidate for LIHC.

Keywords:  ASPM; LIHC; cells proliferation; hepatocellular carcinoma; invasion; m6A; migration

DOI:  https://doi.org/10.1002/jcla.23931
Front Mol Biosci. 2021 ;8 670160

Lipopolysaccharide Alters the m6A Epitranscriptomic Tagging of RNAs in Cardiac Tissue.

Ye-Chen Han, Hong-Zhi Xie, Bo Lu, Ruo-Lan Xiang, Hai-Peng Zhang, Jing-Yi Li, Shu-Yang Zhang.

  N6-methyladenosine (m6A) modification plays important roles in the pathology of a variety of diseases. However, the roles of m6A modification in sepsis-induced myocardial dysfunction are not well defined. Rats were divided into control and lipopolysaccharide (LPS)-induced sepsis group. Global m6A levels of left ventricle tissue were measured by LC-MS/MS, and transcriptome-wide m6A modifications were profiled using epitranscriptomic microarrays (mRNAs and lncRNAs). Bioinformatics analysis was conducted to understand the functional implications of m6A modifications during sepsis. Methylated lncRNAs and mRNAs were measured by m6A single-base site qPCR. The global m6A levels in left ventricle tissue were significantly decreased in the LPS group. While 27 transcripts (23 mRNAs and four lncRNAs) were hypermethylated, 46 transcripts (39 mRNAs and 7 lncRNAs) were hypomethylated in the LPS group. The mRNA expression of writers and readers was significantly decreased in the LPS group. The m6A modification of Clec1b, Stk38l and Tnfrsf26 was associated with platelet activation and apoptotic pathways. Moreover, the decrease in m6A modification of lncRNA XR_346,771 may be related to cation import in cardiac tissue. Our data provide novel information regarding changes to m6A modifications in cardiac tissue during sepsis, and m6A modifications might be promising therapeutic targets.

Keywords:  N6-methyladenosine; epitranscriptomics; lncRNA; myocardial dysfunction; sepsis

DOI:  https://doi.org/10.3389/fmolb.2021.670160
Front Oncol. 2021 ;11 704543

N6-Methyladenosine Regulators and Related LncRNAs Are Potential to be Prognostic Markers for Uveal Melanoma and Indicators of Tumor Microenvironment Remodeling.

Zhicheng Liu, Shanshan Li, Shan Huang, Tao Wang, Zhicheng Liu.

  Uveal melanoma (UM) is one of the most common malignant intraocular tumors in adults. Few studies have investigated the effect of N6-methyladenosine (m6A) RNA methylation regulators and related long noncoding RNAs (lncRNAs) on the tumor microenvironment (TME) and survival time of patients with UM. Based on the transcriptome and clinical data from The Cancer Genome Atlas, we systematically identified m6A regulators. Then, we constructed an m6A regulators-based signature to predict the prognostic risk using univariate and LASSO Cox analyses. The signature was then validated by performing Kaplan-Meier, and receiver operating characteristic analyses. Through the correlation analysis, m6A regulators-related lncRNAs were identified, and they were divided into different clustering subtypes according to their expression. We further assessed differences in TME scores, the survival time of patients, and immune cell infiltration levels between different clustering subtypes. Finally, we screened out the common immune genes shared by m6A-related lncRNAs and determined their expression in different risk groups and clustering subtypes. For further validation, we used single-cell sequencing data from the GSE139829 dataset to explore the expression distribution of immune genes in the TME of UM. We constructed a prognostic risk signature representing an independent prognostic factor for UM using 3 m6A regulators. Patients in the low-risk group exhibited a more favorable prognosis and lower immune cell infiltration levels than patients in the high-risk group. Two subtypes (cluster 1/2) were identified based on m6A regulators-related lncRNAs. The TME scores, prognosis, and immune cell infiltration have a marked difference between cluster 1 and cluster 2. Additionally, 13 common immune genes shared by 5 lncRNAs were screened out. We found that these immune genes were differentially expressed in different risk groups and clustering subtypes and were widely distributed in 3 cell types of TME. In conclusion, our study demonstrated the important role of m6A regulators and related lncRNAs in TME remodeling. The signature developed using m6A regulators might serve as a promising parameter for the clinical prediction of UM.

Keywords:  immune cell infiltration; long noncoding RNAs; m6A RNA methylation regulators; tumor microenvironment; uveal melanoma

DOI:  https://doi.org/10.3389/fonc.2021.704543
Nucleic Acids Res. 2021 Aug 20. pii: gkab714. [Epub ahead of print]

m6A-express: uncovering complex and condition-specific m6A regulation of gene expression.

Teng Zhang, Shao-Wu Zhang, Song-Yao Zhang, Shou-Jiang Gao, Yidong Chen, Yufei Huang.

N6-methyladenosine (m6A) is the most abundant form of mRNA modification and controls many aspects of RNA metabolism including gene expression. However, the mechanisms by which m6A regulates cell- and condition-specific gene expression are still poorly understood, partly due to a lack of tools capable of identifying m6A sites that regulate gene expression under different conditions. Here we develop m6A-express, the first algorithm for predicting condition-specific m6A regulation of gene expression (m6A-reg-exp) from limited methylated RNA immunoprecipitation sequencing (MeRIP-seq) data. Comprehensive evaluations of m6A-express using simulated and real data demonstrated its high prediction specificity and sensitivity. When only a few MeRIP-seq samples may be available for the cellular or treatment conditions, m6A-express is particularly more robust than the log-linear model. Using m6A-express, we reported that m6A writers, METTL3 and METTL14, competitively regulate the transcriptional processes by mediating m6A-reg-exp of different genes in Hela cells. In contrast, METTL3 induces different m6A-reg-exp of a distinct group of genes in HepG2 cells to regulate protein functions and stress-related processes. We further uncovered unique m6A-reg-exp patterns in human brain and intestine tissues, which are enriched in organ-specific processes. This study demonstrates the effectiveness of m6A-express in predicting condition-specific m6A-reg-exp and highlights the complex, condition-specific nature of m6A-regulation of gene expression.

DOI: https://doi.org/10.1093/nar/gkab714
Cell Death Discov. 2021 Aug 14. 7(1): 215

IGF2BP2-modified circular RNA circARHGAP12 promotes cervical cancer progression by interacting m6A/FOXM1 manner.

Fei Ji, Yang Lu, Shaoyun Chen, Yan Yu, Xiaoling Lin, Yuanfang Zhu, Xin Luo.

Emerging evidence indicates that circular RNA (circRNA) and N6-methyladenosine (m6A) play critical roles in cervical cancer. However, the synergistic effect of circRNA and m6A on cervical cancer progression is unclear. In the present study, our sequencing data revealed that a novel m6A-modified circRNA (circARHGAP12, hsa_circ_0000231) upregulated in the cervical cancer tissue and cells. Interestingly, the m6A modification of circARHGAP12 could amplify its enrichment. Functional experiments illustrated that circARHGAP12 promoted the tumor progression of cervical cancer in vivo and vitro. Furthermore, MeRIP-Seq illustrated that there was a remarkable m6A site in FOXM1 mRNA. CircARHGAP12 interacted with m6A reader IGF2BP2 to combine with FOXM1 mRNA, thereby accelerating the stability of FOXM1 mRNA. In conclusion, we found that circARHGAP12 exerted the oncogenic role in cervical cancer progression through m6A-dependent IGF2BP2/FOXM1 pathway. These findings may provide new concepts for cervical cancer biology and pathological physiology.

DOI: https://doi.org/10.1038/s41420-021-00595-w
BMC Cancer. 2021 Aug 20. 21(1): 938

Characteristic of molecular subtypes in lung adenocarcinoma based on m6A RNA methylation modification and immune microenvironment.

Hao Zhou, Miaosen Zheng, Muqi Shi, Jinjie Wang, Zhanghao Huang, Haijian Zhang, Youlang Zhou, Jiahai Shi.

   BACKGROUND: Lung adenocarcinoma (LUAD) is a major subtype of lung cancer and closely associated with poor prognosis. N6-methyladenosine (m6A), one of the most predominant modifications in mRNAs, is found to participate in tumorigenesis. However, the potential function of m6A RNA methylation in the tumor immune microenvironment is still murky.
METHODS: The gene expression profile cohort and its corresponding clinical data of LUAD patients were downloaded from TCGA database and GEO database. Based on the expression of 21 m6A regulators, we identified two distinct subgroups by consensus clustering. The single-sample gene-set enrichment analysis (ssGSEA) algorithm was conducted to quantify the relative abundance of the fraction of 28 immune cell types. The prognostic model was constructed by Lasso Cox regression. Survival analysis and receiver operating characteristic (ROC) curves were used to evaluate the prognostic model.
RESULT: Consensus classification separated the patients into two clusters (clusters 1 and 2). Those patients in cluster 1 showed a better prognosis and were related to higher immune scores and more immune cell infiltration. Subsequently, 457 differentially expressed genes (DEGs) between the two clusters were identified, and then a seven-gene prognostic model was constricted. The survival analysis showed poor prognosis in patients with high-risk score. The ROC curve confirmed the predictive accuracy of this prognostic risk signature. Besides, further analysis indicated that there were significant differences between the high-risk and low-risk groups in stages, status, clustering subtypes, and immunoscore. Low-risk group was related to higher immune score, more immune cell infiltration, and lower clinical stages. Moreover, multivariate analysis revealed that this prognostic model might be a powerful prognostic predictor for LUAD. Ultimately, the efficacy of this prognostic model was successfully validated in several external cohorts (GSE30219, GSE50081 and GSE72094).
CONCLUSION: Our study provides a robust signature for predicting patients' prognosis, which might be helpful for therapeutic strategies discovery of LUAD.

Keywords:  Immune microenvironment; Lung adenocarcinoma; Prognostic model; m6A RNA methylation

DOI:  https://doi.org/10.1186/s12885-021-08655-1
Cancer Cell Int. 2021 Aug 16. 21(1): 432

Identification of a robust signature for clinical outcomes and immunotherapy response in gastric cancer: based on N6-methyladenosine related long noncoding RNAs.

Tenghui Han, Dong Xu, Jun Zhu, Jipeng Li, Lei Liu, Yanchun Deng.

   BACKGROUND: Gastric cancer (GC) is a globally prevalent cancer, ranking fifth for incidence and fourth for mortality worldwide. The N6-methyladenosine (m6A) related long noncoding RNAs (lncRNAs) were widely investigated in recent studies. Nevertheless, the underlying prognostic implication and tumor immune mechanism of m6A-related lncRNA in GC remain unknown.
METHODS: We systematically assessed the m6A modification expression of 407 GC clinical samples based on 23 m6A regulators and comprehensively associated these genes with lncRNAs. Then, we constructed a m6A-related lncRNA prognostic signature (m6A-LPS) to evaluate both status and prognosis of the disease. Immune-related mechanisms were explored via dissecting tumor-infiltrating cells as well as applying tumor immune dysfunction and the exclusion algorithm. Furthermore, we validated the latent regulative mechanism of m6A-related lncRNA in GC cell lines.
RESULTS: The m6A-LPS containing nine hub lncRNAs was built, which possessed a superior capability to predict the outcomes of GC patients. Meanwhile, we found an intimate correlation between the m6A-LPS and tumor infiltrating cells, and that the low-risk group had a higher expression of immune checkpoints and responsed more to immunotherapy than the high-risk group. Clinically, these crucial lncRNAs expression levels were verified in ten pairs of GC samples. In in vitro experiments, the abilities of migration and proliferation were significantly enhanced via downregulating the lncRNA AC026691.1. Both migrative and proliferative capabilities of tumor cells were significantly enhanced via downregulating the lncRNA AC026691.1. in vitro.
CONCLUSIONS: Collectively, the m6A-LPS could provide a novel prediction insight into the prognosis of GC patients and serve as an independent clinical factor for GC. These m6A-related lncRNAs might remodel the tumor microenvironment and affect the anti-cancer ability of immune checkpoint blockers. Importantly, lncRNA AC026691.1 could inhibit both migration and proliferation of GC by means of FTO regulation.

Keywords:  FTO; Gastric cancer; Immune checkpoint blockers; LncRNA; m6A modification

DOI:  https://doi.org/10.1186/s12935-021-02146-w
J Oncol. 2021 ;2021 8875424

m6A Methyltransferase 3 Promotes the Proliferation and Migration of Gastric Cancer Cells through the m6A Modification of YAP1.

Wenjie Zhou, Qingying Xian, Qi Wang, Chen Wu, Haijiao Yan, Xiaodong Li, Lu Lu, Changping Wu, Danxia Zhu, Xiaoli Xu, Jun Wu.

Gastric cancer is the most common gastrointestinal tumor with an increasing incidence. Furthermore, advanced gastric cancer is more common, but the mechanism underlying the proliferation and metastasis of gastric cancer has not been thoroughly explored. N6-methyladenosine (m6A) methyltransferase 3 (METTL3) may be involved in the proliferation and metastasis of gastric cancer. Therefore, Yes-associated protein 1 (YAP1) in the Hippo pathway was selected as the target, and the relationship between METTL3 and the proliferation and metastasis of gastric cancer was proved through a series of experiments. This research showed that the expression of m6A and METTL3 was upregulated in human gastric cancer tissues and gastric cancer cell lines. After lentiviral transfection, METTL3 silencing in AGS (human gastric adenocarcinoma cell line AGS) and MKN-45 (human gastric cancer cell line MKN-45) gastric cancer cell lines directly inhibited the proliferation, aggressiveness, and migration of gastric cancer cells. Mechanically, the inhibition of the YAP1-TEAD signaling pathway by peptide 17 reduces m6A methylation and the total mRNA level of YAP1. It also eliminates the promoting effect of METTL3 on the proliferation and migration of gastric cancer cells. In turn, the overexpression of YAP1 eliminates the inhibitory effect of METTL3 silencing on the proliferation, migration, and invasion of gastric cancer cells. This article proved that m6A methyltransferase METTL3 promoted the proliferation and migration of gastric cancer cells through the m6A modification of YAP1.

DOI: https://doi.org/10.1155/2021/8875424
Sci Adv. 2021 Aug;pii: eabi8215. [Epub ahead of print]7(34):

N6-methyladenosine binding induces a metal-centered rearrangement that activates the human RNA demethylase Alkbh5.

Jeffrey A Purslow, Trang T Nguyen, Balabhadra Khatiwada, Aayushi Singh, Vincenzo Venditti.

Alkbh5 catalyzes demethylation of the N 6-methyladenosine (m6A), an epigenetic mark that controls several physiological processes including carcinogenesis and stem cell differentiation. The activity of Alkbh5 comprises two coupled reactions. The first reaction involves decarboxylation of α-ketoglutarate (αKG) and formation of a Fe4+═O species. This oxyferryl intermediate oxidizes the m6A to reestablish the canonical base. Despite coupling between the two reactions being required for the correct Alkbh5 functioning, the mechanisms linking dioxygen activation to m6A binding are not fully understood. Here, we use solution NMR to investigate the structure and dynamics of apo and holo Alkbh5. We show that binding of m6A to Alkbh5 induces a metal-centered rearrangement of αKG that increases the exposed area of the metal, making it available for binding O2 Our study reveals the molecular mechanisms underlying activation of Alkbh5, therefore opening new perspectives for the design of novel strategies to control gene expression and cancer progression.

DOI: https://doi.org/10.1126/sciadv.abi8215
IUBMB Life. 2021 Aug 18.

m6 A transferase KIAA1429-stabilized LINC00958 accelerates gastric cancer aerobic glycolysis through targeting GLUT1.

Desheng Yang, Shuang Chang, Fuchun Li, Mingxiao Ma, Jiayao Yang, Xun Lv, Luxin Huangfu, Changhe Jia.

  Emerging evidence has demonstrated that N6 -methyladenosine (m6 A) and long noncoding RNAs (lncRNAs) are both crucial regulators in gastric cancer (GC) tumorigenesis. However, the interaction of m6 A and lncRNAs in GC progression are still unclear. Here, our team discovered that lncRNA LINC00958 expression up-regulated in GC tissue and cells. Clinically, high-expression of LINC00958 was clinically correlated to lower survival of GC patients. Functionally, in vitro assays demonstrated that LINC00958 promoted the GC cells' aerobic glycolysis. Mechanistically, methylated RNA immunoprecipitation sequencing (MeRIP-Seq) found that there were m6 A-modificated sites in LINC00958, and moreover m6 A methyltransferase KIAA1429 catalyzed the m6 A modification on LINC00958 loci. Moreover, LINC00958 interacted with GLUT1 mRNA via the m6 A-dependent manner to enhance GLUT1 mRNA transcript stability, thereby positively regulating the aerobic glycolysis of GC. In conclusion, our findings reveal the function and mechanism of KIAA1429-induced LINC00958 in GC, delineating novel understanding of m6 A-lncRNA in cancer biology.

Keywords:  GLUT1; KIAA1429; N6-methyladenosine; aerobic glycolysis; gastric cancer

DOI:  https://doi.org/10.1002/iub.2545
STAR Protoc. 2021 Sep 17. 2(3): 100724

Quantitative analysis of m6A RNA modification by LC-MS.

Lavina Mathur, Sunhee Jung, Cholsoon Jang, Gina Lee.

  N6-adenosine methylation (m6A) of messenger RNA (mRNA) plays key regulatory roles in gene expression. Accurate measurement of m6A levels is thus critical to understand its dynamic changes in various biological settings. Here, we provide a protocol to quantitate the levels of adenosine and m6A in cellular mRNAs. Using nuclease and phosphatase, we digest mRNA into nucleosides, which are subsequently quantified using liquid chromatography mass spectrometry. For complete details on the use and execution of this protocol, please refer to Cho et al. (2021).

Keywords:  cell biology; cell culture; chemistry; mass spectrometry; metabolism; molecular biology

DOI:  https://doi.org/10.1016/j.xpro.2021.100724
Mol Med. 2021 Aug 19. 27(1): 91

Human umbilical cord mesenchymal stem cells deliver exogenous miR-26a-5p via exosomes to inhibit nucleus pulposus cell pyroptosis through METTL14/NLRP3.

Xiaoqiu Yuan, Tiefeng Li, Lei Shi, Jinhao Miao, Yongfei Guo, Yu Chen.

   BACKGROUND: Intervertebral disc degeneration (IVDD) is the breakdown of the discs supporting the vertebrae. It is one of the most frequent causes of back pain worldwide. Currently, the clinical interventions for IVDD are mainly focused on symptom releases. Thus, new therapeutic options are needed.
METHODS: Nucleus pulposus (NP) samples were obtained from 20 patients experiencing IVDD and 10 healthy volunteers compared for mRNA N6-methyladenosine (m6A) mRNA modification as well as methyltransferase (METT) like METTL3, METTL14, and Wilms' tumor 1-associated protein mRNA and protein abundance following exosomes exposure from mesenchymal stem cells. In addition, microRNA expressions were also compared. The correlation between the NLR family pyrin domain containing 3 (NLRP3) and METTL14 was measured by luciferase reporter assay. Cytokines were evaluated using an enzyme-linked immunosorbent assay. METTL14, NLRP3, and insulin-like growth factor 2 mRNA-binding protein 2 mRNAs were measured via a quantitative reverse transcription-polymerase chain reaction. Protein was assayed using western blots. Cell death was assessed by propidium iodide staining, lactate dehydrogenase release, gasdermin-N domain abundance, and caspase-1 activation.
RESULTS: The human umbilical cord mesenchymal stem cell (hucMSC) exosomes were found to effectively improve the viability of NP cells and protect them from pyroptosis through targeting METTL14, with a methyltransferase catalyzing m6A modification. METTL14 was highly present in NP cells from IVDD patients, which stabilize NLRP3 mRNA in an IGFBP2-dependent manner. The elevated NLRP3 levels result in the increase of interleukin 1β (IL-1β) and IL-18 levels and trigger pyroptotic NP cell death. Such pathogenic axis could be blocked by hucMSC exosomes, which directly degrade METTL14 through exosomal miR-26a-5p.
CONCLUSIONS: The results of the current study revealed the beneficial effects of hucMSC exosomes on NP cells and determined a potential mechanism inducing IVDD.

Keywords:  Exosomal miR-26a-5p; Intervertebral disc degeneration; N6-methyladenosine; Pyroptosis; Umbilical cord mesenchymal stem cells

DOI:  https://doi.org/10.1186/s10020-021-00355-7
Oncoimmunology. 2021 ;10(1): 1962656

YTHDF1 and YTHDF2 are associated with better patient survival and an inflamed tumor-immune microenvironment in non-small-cell lung cancer.

Kazuo Tsuchiya, Katsuhiro Yoshimura, Yusuke Inoue, Yuji Iwashita, Hidetaka Yamada, Akikazu Kawase, Takuya Watanabe, Masayuki Tanahashi, Hiroshi Ogawa, Kazuhito Funai, Kazuya Shinmura, Takafumi Suda, Haruhiko Sugimura.

  The human YTH domain family (YTHDF) proteins are RNA-binding proteins that recognize N6-methyladenosine (m6A), facilitating various biological processes via m6A RNA modification. How these molecules associate with non-small-cell lung cancer (NSCLC) molecular mechanisms remain unclear. The protein expression levels of YTHDF1 and YTHDF2 in 603 cases of resected NSCLC were evaluated using immunohistochemistry. We analyzed the associations of these attributes with patient characteristics and survival. We also assessed four subsets of lymphocytes (PD-1+, CD8+, Foxp3+, and CD45RO+) as tumor-infiltrating lymphocytes (TILs) in the tumor nest and in the surrounding stroma separately. In addition, we investigated differentially expressed genes and the expression of PD-L1 in YTHDF1- and YTHDF2-deprived lung cancer cells. The expressions of both YTHDF1 and YTHDF2 were less in the advanced-stage tumors than in the early-stage tumors. The expressions of both YTHDF1 and YTHDF2 were also independent favorable prognostic factors for recurrence-free survival (HR, 0.745; 95% CI, 0.562-0.984 for YTHDF1; HR, 0.683; 95% CI, 0.503-0.928 for YTHDF2). The TIL densities of almost all four lymphocyte subsets in the stroma were significantly higher in the tumors with high YTHDF1 and YTHDF2 expression. In vitro, YTHDF1 and YTHDF2 knockdown in cells upregulated tumor PD-L1 expression and altered multiple immune-related genes. High expressions of both YTHDF1 and YTHDF2 are associated with a favorable prognostic outcome of NSCLC patients, a greater amount of TILs, and downregulation of PD-L1. YTHDF1 and YTHDF2 could be novel prognostic and druggable targets related to the tumor-immune microenvironment in lung cancers.

Keywords:  Lung cancer; m6A; pd-l1; tumor-immune microenvironment; ythdf1; ythdf2

DOI:  https://doi.org/10.1080/2162402X.2021.1962656
PeerJ. 2021 ;9 e11893

Network analysis of miRNA targeting m6A-related genes in patients with esophageal cancer.

Lili Li, Rongrong Xie, Qichun Wei.

   Background: We investigated the miRNA-m6A related gene network and identified a miRNA-based prognostic signature in patients with esophageal cancer using integrated genomic analysis.
Methods: We obtained expression data for m6A-related genes and miRNAs from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) datasets. Survival analysis was conducted to identify potential prognostic biomarkers. LASSO Cox regression was performed to construct the overall survival (OS) associated prediction signature. We used the Kaplan-Meier (K-M) curve and receiver operating characteristic (ROC) curves to explore the signature's efficiency and accuracy. Interactions between the m6A-related genes and miRNAs were identified in starBase3.0 and used to construct the miRNA-m6A related gene network.
Results: We found that HNRNPC, YTHDF, ZC3H13, YTHDC2, and METTL14 were dysregulated in esophageal cancer tissues. Multivariate Cox regression analysis revealed that HNRNPC may be an independent risk factor for OS. Five hundred twenty-two potential upstream miRNAs were obtained from starBase3.0. Four miRNAs (miR-186, miR-320c, miR-320d, and miR-320b) were used to construct a prognostic signature, which could serve as a prognostic predictor independent from routine clinicopathological features. Finally, we constructed a key miRNA-m6A related gene network and used one m6A-related gene and four miRNAs associated with the prognosis. The results of our bioinformatics analysis were successfully validated in the human esophageal carcinoma cell lines KYSE30 and TE-1.
Conclusion: Our study identified a 4-miRNA prognostic signature and established a key miRNA-m6A related gene network. These tools may reliably assist with esophageal cancer patient prognosis.

Keywords:  Esophageal cancer; HNRNPC; N6-methyladenosine; Overall survival; Prognosis

DOI:  https://doi.org/10.7717/peerj.11893
Medicine (Baltimore). 2021 Jul 23. 100(29): e26648

A seven-m6A regulator-related CpG site-based prognostic signature for endometrial carcinoma.

Xiang Zhang, Xuecheng Pang, Yue Huang, Sumin Qian.

BACKGROUND: Endometrial carcinoma (EC) has become a common gynecologic malignancy with a high mortality. The m6A regulators have been identified to be closely associated with multiple human cancers including EC. However, the CpG methylation signature related to m6A regulators in EC remains unclear.
METHOD: The methylation profiles of EC patients including cancer samples and adjacent normal samples were obtained from The Cancer Genome Atlas (TCGA) database. The CpG sites in 20 m6A regulators were identified. Univariate Cox regression and LASSO Cox regression analysis were used to screen key CpG sites which were located at m6A regulators and significantly related to the prognosis of EC. The predictive model for EC prognosis was constructed, and multivariate Cox regression analysis was applied to explore whether the risk score derived from the model could function as an independent signature for EC prognosis. Meanwhile, a nomogram model was constructed by combing the independent prognostic signatures for prediction of the long-term survival in EC patients.
RESULTS: A total of 396 CpG sites located at 20 m6A regulators were identified. A specific predictive model for EC prognosis based on 7 optimal CpG sites was constructed, which presented good performance in prognosis prediction of EC patients. Moreover, risk score was determined to be an independent signature both in the training set and validation set. By bringing in three independent prognostic factors (age, risk score, and TNM stage), the nomogram was constructed and could effectively predict the 3- and 5-year survival rates of EC patients.
CONCLUSION: Our study suggested that the CpG sites located at m6A regulators might be considered as potential prognostic signatures for EC patients.

DOI: https://doi.org/10.1097/MD.0000000000026648
Immunol Rev. 2021 Aug 17.

How RNA modifications regulate the antiviral response.

Matthew G Thompson, Matthew T Sacco, Stacy M Horner.

  Induction of the antiviral innate immune response is highly regulated at the RNA level, particularly by RNA modifications. Recent discoveries have revealed how RNA modifications play key roles in cellular surveillance of nucleic acids and in controlling gene expression in response to viral infection. These modifications have emerged as being essential for a functional antiviral response and maintaining cellular homeostasis. In this review, we will highlight these and other discoveries that describe how the antiviral response is controlled by modifications to both viral and cellular RNA, focusing on how mRNA cap modifications, N6-methyladenosine, and RNA editing all contribute to coordinating an efficient response that properly controls viral infection.

Keywords:  N6-methyladenosine; RNA editing; adenosine deaminases acting on RNA; cap modification; innate immunity; interferon; pattern recognition receptors

DOI:  https://doi.org/10.1111/imr.13020
J Oncol. 2021 ;2021 6173206

m5C-Related Signatures for Predicting Prognosis in Cutaneous Melanoma with Machine Learning.

Maoxin Huang, Yi Zhang, Xiaohong Ou, Caiyun Wang, Xueqing Wang, Bibo Qin, Qiong Zhang, Jie Yu, Jianxiang Zhang, Jianbin Yu.

Background: Cutaneous melanoma (CM) is one of the most life-threatening primary skin cancers and is prone to distant metastases. A widespread presence of posttranscriptional modification of RNA, 5-methylcytosine (m5C), has been observed in human cancers. However, the potential mechanism of the tumorigenesis and prognosis in CM by dysregulated m5C-related regulators is obscure.
Methods: We use comprehensive bioinformatics analyses to explore the expression of m5C regulators in CM, the prognostic implications of the m5C regulators, the frequency of the copy number variant (CNV), and somatic mutations in m5C regulators. Additionally, the CM patients were divided into three clusters for better predicting clinical features and outcomes via consensus clustering of m5C regulators. Then, the risk score was established via Lasso Cox regression analysis. Next, the prognosis value and clinical characteristics of m5C-related signatures were further explored. Then, machine learning was used to recognize the outstanding m5C regulators to risk score. Finally, the expression level and clinical value of USUN6 were analyzed via the tissue microarray (TMA) cohort.
Results: We found that m5C regulators were dysregulated in CM, with a high frequency of somatic mutations and CNV alterations of the m5C regulatory gene in CM. Furthermore, 16 m5C-related proteins interacted with each other frequently, and we divided CM patients into three clusters to better predicting clinical features and outcomes. Then, five m5C regulators were selected as a risk score based on the LASSO model. The XGBoost algorithm recognized that NOP2 and NSUN6 were the most significant risk score contributors. Immunohistochemistry has verified that low expression of USUN6 was closely correlated with CM progression.
Conclusion: The m5C-related signatures can be used as new prognostic biomarkers and therapeutic targets for CM, and NSUN6 might play a vital role in tumorigenesis and malignant progression.

DOI: https://doi.org/10.1155/2021/6173206
Front Cell Dev Biol. 2021 ;9 704341

NAT10-Mediated N4-Acetylcytidine of RNA Contributes to Post-transcriptional Regulation of Mouse Oocyte Maturation in vitro.

Yuting Xiang, Chuanchuan Zhou, Yanyan Zeng, Qi Guo, Jiana Huang, Taibao Wu, Jiawen Liu, Qiqi Liang, Haitao Zeng, Xiaoyan Liang.

  N4-acetylcytidine (ac4C), a newly identified epigenetic modification within mRNA, has been characterized as a crucial regulator of mRNA stability and translation efficiency. However, the role of ac4C during oocyte maturation, the process mainly controlled via post-transcriptional mechanisms, has not been explored. N-acetyltransferase 10 (NAT10) is the only known enzyme responsible for ac4C production in mammals and ac4C-binding proteins have not been reported yet. In this study, we have documented decreasing trends of both ac4C and NAT10 expression from immature to mature mouse oocytes. With NAT10 knockdown mediated by small interfering RNA (siRNA) in germinal vesicle (GV)-stage oocytes, ac4C modification was reduced and meiotic maturation in vitro was significantly retarded. Specifically, the rate of first polar body extrusion was significantly decreased with NAT10 knockdown (34.6%) compared to control oocytes without transfection (74.6%) and oocytes transfected with negative control siRNA (72.6%) (p < 0.001), while rates of germinal vesicle breakdown (GVBD) were not significantly different (p = 0.6531). RNA immunoprecipitation and high-throughput sequencing using HEK293T cells revealed that the modulated genes were enriched in biological processes associated with nucleosome assembly, chromatin silencing, chromatin modification and cytoskeletal anchoring. In addition, we identified TBL3 as a potential ac4C-binding protein by a bioinformatics algorithm and RNA pulldown with HEK293T cells, which may mediate downstream cellular activities. Taken together, our results suggest that NAT10-mediated ac4C modification is an important regulatory factor during oocyte maturation in vitro and TBL3 is a potential ac4C-binding protein.

Keywords:  IVM; N4-acetylcytidine; NAT10; RNA modification; oocyte; post-transcriptional modulation

DOI:  https://doi.org/10.3389/fcell.2021.704341