bims-rimeca 2022-10-09 papers

bims-rimeca

Biomed News

on RNA methylation in cancer

Issue of 2022–10–09
28 papers selected by
Sk Ramiz Islam, Saha Institute of Nuclear Physics

Inhibition of the m6A Methyltransferase METTL3 Attenuates the Inflammatory Response in Fusarium solani-Induced Keratitis via the NF-κB Signaling Pathway.
m6A RNA methylation regulates the transcription factors JUN and JUNB in TGF-β-induced epithelial-mesenchymal transition of lung cancer cells.
Targeting N6-methyladenosine RNA modification combined with immune checkpoint Inhibitors: A new approach for cancer therapy.
Alterations of m6A RNA methylation regulators contribute to autophagy and immune infiltration in primary Sjögren's syndrome.
Epitranscriptome profiling of spleen mRNA m6A methylation reveals pathways of host responses to malaria parasite infection.
N6-methyladenosine-related single-nucleotide polymorphism analyses identify oncogene RNFT2 in bladder cancer.
The RNA m6A writer WTAP in diseases: structure, roles, and mechanisms.
Integrated Analyses of m6A Regulator-Based Signature on Its Clinical Application and Immunogenomic Landscape in Stomach Adenocarcinoma.
RNA m6A modifications and potential targeted therapeutic strategies in kidney disease.
METTL3/LINC00662/miR-186-5p feedback loop regulates docetaxel resistance in triple negative breast cancer.
Comprehensive analysis of m6A-modified circRNAs in peritoneal metastasis of high grade serious carcinoma of ovary.
Autophagy induction promoted by m6A reader YTHDF3 through translation upregulation of FOXO3 mRNA.
RNA m6A modifications in mammalian gametogenesis and pregnancy.
Systematic Analysis of Tumor Microenvironment Patterns and Oxidative Stress Characteristics of Endometrial Carcinoma Mediated by 5-Methylcytosine Regulators.
[Research Progress of m6A Methylation Modification in Hematological Tumors--Review].
Elucidation of the substrate of tRNA-modifying enzymes MnmEG leads to in vitro reconstitution of an evolutionarily conserved uridine hypermodification.
Role of 5-methylcytosine in determining the prognosis, tumor microenvironment, and applicability of precision medicine in patients with hepatocellular carcinoma.
High expression of HNRNPR in ESCA combined with 18F-FDG PET/CT metabolic parameters are novel biomarkers for preoperative diagnosis of ESCA.
NSUN2-mediated mRNA m5C Modification Regulates the Progression of Hepatocellular Carcinoma.
Identification of an m6A-related lncRNA prognostic signature in colorectal cancer.
Loss of LOXL2 promotes uterine hypertrophy and tumor progression by enhancing H3K36ac-dependent gene expression.
Nucleophosmin 1 is a prognostic marker of gastrointestinal cancer and is associated with m6A and cuproptosis.
m6A methylation regulators as predictors for treatment of advanced urothelial carcinoma with anti-PDL1 agent.
N6-methyladenosine long non-coding RNAs reveal novel tool to implicate overall survival and immune microenvironment in renal clear cell carcinoma.
Upregulation of CRABP2 by TET1-mediated DNA hydroxymethylation attenuates mitochondrial apoptosis and promotes oxaliplatin resistance in gastric cancer.
IMP1/IGF2BP1 in human colorectal cancer extracellular vesicles.
Chemical Probe-Based Nanopore Sequencing to Selectively Assess the RNA Modifications.
Comprehensive analysis of m6A methylation modification in chronic spinal cord injury in mice.

Invest Ophthalmol Vis Sci. 2022 Oct 03. 63(11): 2

Inhibition of the m6A Methyltransferase METTL3 Attenuates the Inflammatory Response in Fusarium solani-Induced Keratitis via the NF-κB Signaling Pathway.

Hanfeng Tang, Liwei Huang, Jianzhang Hu.

Purpose: The purpose of this study was to elucidate the effect of methyltransferase-like enzyme 3 (METTL3) on inflammation and the NF-κB signaling pathway in fungal keratitis (FK).
Methods: We established corneal stromal cell models and FK mouse models by incubation with Fusarium solani. The overall RNA N6-methyladenosine (m6A) level was determined using an m6A RNA methylation assay kit. The expression of METTL3 was quantified via real-time quantitative polymerase chain reaction (RT-PCR), Western blotting, and immunofluorescence. Subsequently, the level of tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) was identified by Western blotting and immunofluorescence. Moreover, we assessed the effect of METTL3 by transfecting cells with siRNA (in vitro) or adeno-associated virus (in vivo). Hematoxylin and eosin (H&E) staining and slit-lamp biomicroscopy were performed to evaluate corneal damage. Furthermore, the state of NF-κB signaling pathway activation was examined by Western blotting. In addition, RT-PCR and enzyme-linked immunosorbent assays (ELISAs) were performed to evaluate levels of the pro-inflammatory factors interleukin-1β (IL-1β), interleukin-6 (IL-6) and TNF-ɑ.
Results: Our data demonstrated that the levels of the RNA m6A methylation and METTL3 were dramatically increased and that the NF-κB signaling pathway was activated in Fusarium solani-induced keratitis. Inhibition of METTL3 decreased the level of TRAF6, downregulated the phospho-p65(p-p65)/p65 and phospho-IκB(p-IκB)/IκB protein ratios, simultaneously attenuating the inflammatory response and fungal burden in FK.
Conclusions: Our research suggests that the m6A methyltransferase METTL3 regulates the inflammatory response in FK by modulating the NF-κB signaling pathway.

DOI: https://doi.org/10.1167/iovs.63.11.2
J Biol Chem. 2022 Sep 29. pii: S0021-9258(22)00998-X. [Epub ahead of print] 102554

m6A RNA methylation regulates the transcription factors JUN and JUNB in TGF-β-induced epithelial-mesenchymal transition of lung cancer cells.

Kusuma Suphakhong, Minoru Terashima, Sasithorn Wanna-Udom, Risa Takatsuka, Akihiko Ishimura, Takahisa Takino, Takeshi Suzuki.

  N6-methyladenosine (m6A) is the most common internal chemical modification of mRNAs involved in many pathological processes including various cancers. In this study, we investigated the m6A-dependent regulation of JUN and JUNB transcription factors (TFs) during TGF-β-induced epithelial-mesenchymal transition (EMT) of A549 and LC2/ad lung cancer cell lines, as the function and regulation of these TFs within this process remains to be clarified. We found that JUN and JUNB played an important and non-redundant role in the EMT-inducing gene expression program by regulating different mesenchymal genes and that their expressions were controlled by METTL3 m6A methyltransferase. METTL3-mediated regulation of JUN expression is associated with the translation process of JUN protein but not with the stability of JUN protein or mRNA, which is in contrast with the result of m6A-mediated regulation of JUNB mRNA stability. We identified the specific m6A motifs responsible for the regulation of JUN and JUNB in EMT within 3'UTR of JUN and JUNB. Furthermore, we discovered that different m6A reader proteins interacted with JUN and JUNB mRNA and controlled m6A-dependent expression of JUN protein and JUNB mRNA. These results demonstrate that the different modes of m6A-mediated regulation of JUN and JUNB TFs provide critical input in the gene regulatory network during TGF-β-induced EMT of lung cancer cells.

Keywords:  RNA methylation; cancer biology; epithelial-mesenchymal transition; gene expression; m6A reader protein; mRNA stability; translation regulation

DOI:  https://doi.org/10.1016/j.jbc.2022.102554
Comput Struct Biotechnol J. 2022 ;20 5150-5161

Targeting N6-methyladenosine RNA modification combined with immune checkpoint Inhibitors: A new approach for cancer therapy.

Weiwei Liu, Chaoqun Liu, Hui Wang, Lijun Xu, Jueyu Zhou, Sihua Li, Yu Cheng, Rui Zhou, Liang Zhao.

  Immune checkpoint inhibitors (ICIs) have revolutionized cancer immunotherapy by restoring the host antitumor immune response. Since 2011, various ICIs have been approved for the treatment of cancers, which has led to unprecedented prolongation of the survival time for some patients. Although ICIs have been successfully applied in the treatment of different cancers, the low effectiveness rate has dramatically restrained the clinical application of ICI treatment. N6-methyladenosine (m6A) modification is the most common RNA methylation. Recent studies have pointed out that m6A epigenetic modification could improve the efficacy of ICI blockade treatment. Here, we briefly summarize the relevant mechanisms of tumour immunity, the clinical application of ICIs, the resistance to ICI treatment in cancers, and the m6A epigenetic modification and how it regulates the response to ICI treatment. We attempted to provide a potential strategy for cancer therapy by targeting m6A modification combined with ICI blockade treatment.

Keywords:  Cancer immunotherapy; ICB; M6A modification; m6A regulators

DOI:  https://doi.org/10.1016/j.csbj.2022.09.017
Front Immunol. 2022 ;13 949206

Alterations of m6A RNA methylation regulators contribute to autophagy and immune infiltration in primary Sjögren's syndrome.

Linlin Cheng, Haolong Li, Haoting Zhan, Yongmei Liu, Xiaomeng Li, Yuan Huang, Li Wang, Fengchun Zhang, Yongzhe Li.

  N6-methyladenosine (m6A) RNA modification is a new epigenetic regulation mechanism on eukaryotic mRNA. Few autoimmune diseases focused on the role of m6A in their pathogenies, and m6A modulation in the pathological process of primary Sjögren's syndrome (pSS) is still unknown. In this work, three microarray datasets of pSS patients were downloaded from the GEO database: datasets #1 and #2 from the whole peripheral blood (PB) samples, dataset #3 from the labial salivary gland tissue samples, as well as a PB cohort collected from our hospital. Six differentially expressed m6A regulators were identified by comparing the PB dataset #1 of pSS and healthy controls using the Wilcox test and logistic regression analysis. Among them, four (ALKBH5, RBMX, RBM15B, and YTHDF1) were confirmed as down-regulated in PB dataset #2 and in our PB cohort by RT-PCR, and four (ALKBH5, METTL3, RBM15B, and YTHDF1) were confirmed as down-regulated in the dataset #3 of the labial gland tissue. In addition, discrepantly expressed m6A regulators accompanied by diverse immunocytes, including dendritic cells (DCs), T cells, and CD56dim natural killer cells, and among the regulators, ALKBH5 and METTL3 were comprehensively linked with the infiltrated immune cells. Notably, the most enriched autophagy mechanism mediated by m6A was observed in pSS using functional annotation analysis. Ten hub genes were identified using a protein-protein interaction network, and their expression in PB dataset #2 and the expression of three genes (PIK3CA, STAT1, and MAPK3) in the labial gland tissue dataset #3 were confirmed. Our study provides evidence that m6A methylation is widely involved in the immune infiltration and autophagy of pSS, thus contributing to the pathogenesis of this disease and potentially representing a novel therapeutic target.

Keywords:  GEO; autophagy; immune microenvironment; m6A; primary Sjögren’s syndrome

DOI:  https://doi.org/10.3389/fimmu.2022.949206
Front Immunol. 2022 ;13 998756

Epitranscriptome profiling of spleen mRNA m6A methylation reveals pathways of host responses to malaria parasite infection.

Luoluo Wang, Jian Wu, Runzhou Liu, Wenjun Chen, Zhichang Pang, Fan Zhou, Lu Xia, Jia Huang, Tao Pan, Xin-Zhuan Su, Xiaoyun Wang.

  N6 -Methyladenosine (m6A), the most abundant mammalian mRNA modification, has been reported to modulate various viral infections. Although it has been confirmed that RNA modifications can also modulate the replication and development of different parasites, the role of the RNA epitranscriptome in the regulation of host response post parasite infection remains to be elucidated. Here we report host spleen m6A epitranscriptome landscapes induced by different strains of the malaria parasite Plasmodium yoelii. We found that malaria parasite infection dramatically changes host spleen m6A mRNA modification and gene expression. Additionally, malaria parasite infection reprograms host immune response pathways by regulating the m6A modification enzymes. Collectively, our study is the first characterization of host spleen m6A methylome triggered by malaria parasite infections, and our data identify m6A modifications as significant transcriptome-wide marks during host-parasite interactions. We demonstrate that host mRNA methylation machinery can sense and respond to malaria parasite infections, and provide new insights into epitranscriptomic mechanisms underlying parasite-induced pathogenesis.

Keywords:  N6-methyladenosine; RNA epitranscriptome; immune response; malaria infection; plasmodium yoelii

DOI:  https://doi.org/10.3389/fimmu.2022.998756
Cancer Cell Int. 2022 Oct 05. 22(1): 301

N6-methyladenosine-related single-nucleotide polymorphism analyses identify oncogene RNFT2 in bladder cancer.

Jiancheng Lv, Qiang Song, Kexin Bai, Jie Han, Hao Yu, Kai Li, Juntao Zhuang, Xiao Yang, Haiwei Yang, Qiang Lu.

   BACKGROUND: Single-nucleotide polymorphisms (SNPs) in N6-methyladenosine (m6A) related genetic locus play significant roles in tumorigenesis and development. The expression level of many oncogenes and tumour suppressor genes changed because of m6A-associated SNPs. In addition, the relationship between m6A-SNP and bladder cancer (BCa) has not been well studied.
METHODS: We screened m6A-SNPs in BCa by combining m6A-SNPs data and GWAS-SNPs data. Expression quantitative trait loci (eQTL) and differential expression gene (DEGs) analyses were performed. In ring finger protein, transmembrane 2 (RNFT2), rs3088107 (C > G) was found to have significant eQTL signals and make RNFT2 gene differentially-regulated mostly in BCa. We validated the expression level of RNFT2 in 32 pairs of BCa tissues and eight BCa cell lines by quantitative real-time PCR (qRT-PCR). Functional assays were performed to investigate the role of rs3088107 and RNFT2 in BCa in vitro.
RESULTS: We identified 673 m6A-SNPs, which were associated with BCa. Of these m6A-SNPs, 221 showed eQTL signals, amongst which, rs3088107 in RNFT2 showed significant eQTL signals. Results of bioinformatic analyses showed that 11 genes with m6A-SNPs had a differential expression level in BCa. RNFT2 was predicted to be significantly up-regulated in BCa. The qRT-PCR results validated that RNFT2 was highly expressed in our own BCa tissues and cell lines. High expression of RNFT2 also indicated a worse overall survival. We also revealed that rs3088107 (C > G) could inhibit the expression and m6A modification of RNFT2 by qRT-PCR, western-blot and m6A-RIP assays. Moreover, the results of functional assays indicated that RNFT2 promoted BCa cell proliferation and migration.
CONCLUSION: This research found that m6A-SNPs were associated with oncogene RNFT2 in BCa. Furthermore, m6A-SNPs showed great application potential as a new BCa diagnostic biomarker and prognostic indicator.

Keywords:  Bladder cancer; Migration; N6-methyladenosine; Proliferation; Ring finger protein; Single-nucleotide polymorphisms; Transmembrane 2

DOI:  https://doi.org/10.1186/s12935-022-02701-z
Cell Death Dis. 2022 Oct 07. 13(10): 852

The RNA m6A writer WTAP in diseases: structure, roles, and mechanisms.

Qibo Huang, Jie Mo, Zhibin Liao, Xiaoping Chen, Bixiang Zhang.

N6-methyladenosine (m6A) is a widely investigated RNA modification in studies on the "epigenetic regulation" of mRNAs that is ubiquitously present in eukaryotes. Abnormal changes in m6A levels are closely related to the regulation of RNA metabolism, heat shock stress, tumor occurrence, and development. m6A modifications are catalyzed by the m6A writer complex, which contains RNA methyltransferase-like 3 (METTL3), methyltransferase-like 14 (METTL14), Wilms tumor 1-associated protein (WTAP), and other proteins with methyltransferase (MTase) capability, such as RNA-binding motif protein 15 (RBM15), KIAA1429 and zinc finger CCCH-type containing 13 (ZC3H13). Although METTL3 is the main catalytic subunit, WTAP is a regulatory subunit whose function is to recruit the m6A methyltransferase complex to the target mRNA. Specifically, WTAP is required for the accumulation of METTL3 and METTL14 in nuclear speckles. In this paper, we briefly introduce the molecular mechanism of m6A modification. Then, we focus on WTAP, a component of the m6A methyltransferase complex, and introduce its structure, localization, and physiological functions. Finally, we describe its roles and mechanisms in cancer.

DOI: https://doi.org/10.1038/s41419-022-05268-9
Biomed Res Int. 2022 ;2022 2053719

Integrated Analyses of m6A Regulator-Based Signature on Its Clinical Application and Immunogenomic Landscape in Stomach Adenocarcinoma.

Lan Zhang, Qing Zhang, Zebo Huang, Mingxia Zhu, Tongshan Wang, Wei Zhu, Xiaping Wang, Xin Zhou.

Background: The whole tumor microenvironment (TME) infiltration features monitored by integrated roles of different RNA N6-methyladenosine (m6A) regulators remain elusive. Our study is aimed at exploring the association between m6A modification patterns, TME cell-infiltrating levels, and patients' prognosis in stomach adenocarcinoma (STAD) patients.
Methods: Consensus clustering was performed based on the integrated analyses of 17 m6A regulators and 229 m6A-related hallmark genes in STAD (The Cancer Genome Atlas (TCGA) cohort, n = 443; Gene Expression Omnibus (GEO) GSE57303, n = 70, GSE62254 n = 300, and GSE84437 n = 433). A m6ASig scoring system was calculated by the principal component analysis (PCA), and its prognostic value was validated in an independent dataset GES15459.
Results: Three m6A clusters were identified among 1246 STAD patients, which had significant overall survival (OS) differences and demonstrated different TME immune cell infiltration and biological behaviors. According to the m6ASig score, which was generated from the m6A-related hallmark genes, STAD patients were divided into the high-m6ASig group (n = 585) and low-m6ASig group (n = 586). Patients in the high-m6ASig group had a notably prolonged OS and higher immune cell infiltration. Moreover, patients with higher m6ASig score were associated with higher microsatellite instability (MSI); higher PD-L1, CTLA4, and ERBB2 expressions; and greater tumor mutation burden (TMB). Patients with higher m6ASig score demonstrated a better immune response and drug sensitivity.
Conclusion: Our m6ASig scoring system could characterize TME immune cell infiltration, thus predict patient's prognosis and immunotherapy and chemotherapy efficacy, offering a novel tool for the individualized therapeutic implications for STAD patients.

DOI: https://doi.org/10.1155/2022/2053719
Br J Pharmacol. 2022 Oct 04.

RNA m6A modifications and potential targeted therapeutic strategies in kidney disease.

Wei-Jian Ni, Hao Lu, Nan-Nan Ma, Bing-Bing Hou, Jing Zeng, Hong Zhou, Wei Shao, Xiao-Ming Meng.

  Epigenetic modifications have received increasing attention and have been shown to be extensively involved in kidney development and disease progression. Among them, the most common RNA modification, N6-methyladenosine (m6A), has been shown to dynamically and reversibly exert its functions in multiple ways, including splicing, export, decay, and translation initiation efficiency, to regulate mRNA fate. Moreover, m6A has also been reported to exert biological effects by destabilizing base pairing to modulate various functions of RNAs. Most importantly, an increasing number of kidney diseases, such as renal cell carcinoma, acute kidney injury and chronic kidney disease, have been found to be associated with aberrant m6A patterns. In this review, we comprehensively review the critical roles of m6A in kidney diseases and discuss the possibilities and relevance of m6A-targeted epigenetic therapy, with an integrated comprehensive description of the detailed alterations in specific loci that contribute to cellular processes that are associated with kidney diseases.

Keywords:  N6-methyladenosine; RNA epigenetic modification; acute kidney injury; chronic kidney disease; renal cell carcinoma; targeted therapy

DOI:  https://doi.org/10.1111/bph.15968
Sci Rep. 2022 Oct 06. 12(1): 16715

METTL3/LINC00662/miR-186-5p feedback loop regulates docetaxel resistance in triple negative breast cancer.

Lei Jing, Liu Lan, Zhang Mingxin, Zhang Zhaofeng.

Insight into the mechanism of docetaxel resistance in breast cancer may help to improve prognosis. We aimed to investigate the role of N6-methyladenosine (m6A) and the METTL3/LINC00662/miR-186-5p pathway in regulating docetaxel resistance in triple negative breast cancer (TNBC). We have recruited 193 pathologically diagnosed TNBC patients from 2016 to 2017 in our hospital. Quantitative real-time PCR was used to evaluate the expression of LINC00662 and miR-186-5p both in vivo and in vitro. CCK8 tests were used to assess cell viability. ELISA was used for protein expression evaluation. Dual luciferase reporter gene assay and RNA pull-down were used to evaluate the interaction between LINC00662 and miR-186-5p. m6A levels were enhanced in breast cancer tissues and cells. LINC00662, miR-186-5p and METTL3 were differentially expressed in vivo, and METTL3 expression was associated with LINC00662 and miR-186-5p expression. LINC00662 and miR-186-5p were differentially expressed in vitro; LINC00662 promoted cell viability and decreased the apoptosis rate, whereas miR-186-5p inhibited cell viability and increased the apoptosis rate. Furthermore, we found that METTL3 regulated m6A levels in docetaxel-resistant breast cancer cells by regulating the expression of LINC00662. Moreover, LINC00662 and miR-186-5p regulated the cell viability rate of docetaxel-resistant breast cancer cells. Further experiments showed that LINC00662 directly interacted with miR-186-5p to exert biological functions; besides miR-186-5p could regulate the expression of METTL3. METTL3 promotes m6A levels and docetaxel resistance in breast cancer by regulating the expression of LINC00662 and miR-186-5p; more experiments are needed to clarify the role of m6A regulation in drug resistance.

DOI: https://doi.org/10.1038/s41598-022-20477-0
Front Oncol. 2022 ;12 988578

Comprehensive analysis of m6A-modified circRNAs in peritoneal metastasis of high grade serious carcinoma of ovary.

Lin Guo, Nini Xu, Daner Qiu, Xiaozhe Yang, Shasha Zhao, Hongxi Zhao.

   Purpose: High-grade serous ovarian cancer (HGSOC) remains the most lethal female cancer due to metastasis. CircRNAs are recently identified to be modified by N6-methyladenosine (m6A) in many cells. However, the significance of m6A-modified circular RNAs (circRNAs) has not been elucidated in HGSOC peritoneal metastasis. Here, we aimed to investigate the participation and potential functions of m6A-modified circRNAs in HGSCO peritoneal metastasis.
Methods: Cancerous tissues were collected from the in situ and the peritoneal metastasis lesions of HGSCO patients. M6A-tagged circRNAs were identified by m6A-modified RNA immunoprecipitation sequencing (m6A-RIP-seq). Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed to predict the potential functions of the m6A-modified circRNAs.
Results: For the m6A-modified circRNAs, 259 were upregulated and 227 were downregulated in the peritoneal metastasis than in the situ lesions of HGSCO patients. For the m6A peaks, 1541 were upregulated and 1293 were downregulated in the peritoneal metastasis than in the in situ lesions of HGSCO patients. For the differential expressed circRNAs, 1911(19.6%) were upregulated and 2883(29.6%) were downregulated in the peritoneal metastasis than in the in situ lesions of HGSCO patients. The upregulated m6A-modified circRNAs were associated with the HIF-1 signaling. The downregulated m6A-modified circRNAs were associated with the MAPK signaling.
Conclusions: This work firstly identified the transcriptome-wide map of m6A-modified circRNAs in peritoneal metastasis of HGSCO. Our findings provided novel evidences about the participation of m6A-modified circRNAs via HIF-1 and MAPK signaling and a new insight in molecular target of HGSCO peritoneal metastasis.

Keywords:  HGSOC; N6-methyladenosine; circular RNA; high grade serious carcinoma of ovary peritoneal metastasis; m6A-modified circRNAs

DOI:  https://doi.org/10.3389/fonc.2022.988578
Nat Commun. 2022 Oct 04. 13(1): 5845

Autophagy induction promoted by m6A reader YTHDF3 through translation upregulation of FOXO3 mRNA.

WeiChao Hao, MeiJuan Dian, Ying Zhou, QiuLing Zhong, WenQian Pang, ZiJian Li, YaYan Zhao, JiaCheng Ma, XiaoLin Lin, RenRu Luo, YongLong Li, JunShuang Jia, HongFen Shen, ShiHao Huang, GuanQi Dai, JiaHong Wang, Yan Sun, Dong Xiao.

Autophagy is crucial for maintaining cellular energy homeostasis and for cells to adapt to nutrient deficiency, and nutrient sensors regulating autophagy have been reported previously. However, the role of eiptranscriptomic modifications such as m6A in the regulation of starvation-induced autophagy is unclear. Here, we show that the m6A reader YTHDF3 is essential for autophagy induction. m6A modification is up-regulated to promote autophagosome formation and lysosomal degradation upon nutrient deficiency. METTL3 depletion leads to a loss of functional m6A modification and inhibits YTHDF3-mediated autophagy flux. YTHDF3 promotes autophagy by recognizing m6A modification sites around the stop codon of FOXO3 mRNA. YTHDF3 also recruits eIF3a and eIF4B to facilitate FOXO3 translation, subsequently initiating autophagy. Overall, our study demonstrates that the epitranscriptome regulator YTHDF3 functions as a nutrient responder, providing a glimpse into the post-transcriptional RNA modifications that regulate metabolic homeostasis.

DOI: https://doi.org/10.1038/s41467-022-32963-0
Reproduction. 2022 Oct 01. pii: REP-22-0112. [Epub ahead of print]

RNA m6A modifications in mammalian gametogenesis and pregnancy.

Xuesong Sui, Arne Klungland, Lu Gao.

The epitranscriptome is defined as the collection of post-transcriptional chemical modifications of RNA in a cell. RNA methylation refers to the chemical post-transcriptional modification of RNA by selectively adding methyl groups under the catalysis of a methyltransferase. The N-6 methyladenosine (m6A) is one of the most common of the more than 100 known RNA modifications. Recent research has revealed that RNA m6A modifications are reversible. Additionally, m6A containing RNA can be selectively identified by immunoprecipitation followed by high-throughput sequencing (MeRIP-SEQ). These two developments have inspired a tremendous effort to unravel the biological role of m6A. The role of RNA m6A modifications in immune regulation, cell division, stem cell renewal, gametogenesis, embryonic development, and placental function have gradually emerged, which is of great significance for the study of post-transcriptional regulation of gene expression in reproductive biology. This review summarizes the current knowledge about RNA m6A modification in a variety of mammalian reproductive events.

DOI: https://doi.org/10.1530/REP-22-0112
Oxid Med Cell Longev. 2022 ;2022 6431164

Systematic Analysis of Tumor Microenvironment Patterns and Oxidative Stress Characteristics of Endometrial Carcinoma Mediated by 5-Methylcytosine Regulators.

Chunli Dong, Ling Dang, Xiaocui Gao, Renyan Xu, Hui Zhang, Xin Zhang.

As a widely distributed RNA methylation modification, m5C is involved in the regulation of tumorigenesis. Nevertheless, its fundamental process is not clear. This research sought to examine the genetic properties of the 5-methylcytosine (m5C) regulator in endometrial carcinoma, as well as the prognostic significance and impact of m5C regulators on oxidative stress. Therefore, the TCGA-UCEC data set was used to explore the characteristics of 17 RNAm5C-related genes in the transcriptome, genome, and regulatory network. The subtypes of RNAm5C in UCEC were identified based on the expression levels of 17 RNAm5C-related genes. The prognosis of RNAm5C-2 was significantly better than that of RNAm5C-1. Then, we examined the differences (variations) across various subtypes in terms of immune cell infiltration (ICI) as well as the expression of immune-related signal markers. The findings demonstrated that there were distinct variations in the infiltration level of immune cells in each subtype, which may be the reason for the differences in the prognosis of each subtype. In addition, the differentially expressed genes (DEGs) among RNAm5C subtypes of different UCEC tumors were identified, and the DEGs significant for survival were screened. After obtaining 34 prognostic genes, the dimensionality was reduced to construct an RNA methylation score (RS). As per the findings, RS is a more accurate marker for determining the prognosis for patients with endometrial cancer. The RS was used to categorize UCEC tumor samples, and these results led to the formation of high-score and low-score groups. The patients in the group with a high-RNA methylation score exhibited a survival time that was considerably longer in contrast with those in the group with a low-RNA methylation score. The capacity of RS to predict whether or not immunotherapy would be beneficial was explored further. In the group with a high-RNA methylation score, the objective response rate to the anti-PD-L1 therapy was substantially greater compared to that observed in the subgroup with a low-RNA methylation score. Additionally, there were variations across various RS groups in terms of clinical features, tumor mutation burden, and the infiltration level of immune cells. After binary tree analysis and PCR verification of 34 prognostic genes, it is finally found that the six genes of MAGOH3P, TRBJ2_3, YTHDF1P1, RP11_323D18.5, RP11_405M12.2, and ADAM30 are significantly overexpressed in cancer tissues. These genes can be used as potential biomarkers of endometrial cancer and provide data support for precise immunotherapy in UCEC tumors.

DOI: https://doi.org/10.1155/2022/6431164
Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2022 Oct;30(5): 1607-1611

[Research Progress of m6A Methylation Modification in Hematological Tumors--Review].

Feng Li, Fei-Fei Yang, Yan-Li Xu.

  N6-methyladenosine (m6A) is one of the most common epigenetic modifications of eukaryotic mRNAs, which is involved in the regulation of gene expressions and biological processes in a variety of cells with dynamic and reversible methylation processes. In recent years, many studies have shown that m6A methylation modification not only acts on the growth, proliferation, and medullary differentiation of acute myeloid leukemia cells, but also participates in the regulation of the proliferation and apoptosis of other hematological tumor cells such as chronic myeloid leukemia and diffuse large B-cell lymphoma, and it can even weaken the efficacy of anti-hematological tumor immunotherapy and induce immune escape leading to tumor resistance. With the successive development of a variety of m6A methylation-related enzyme inhibitors, it will provide new therapeutic ideas for patients with relapsed and refractory hematological tumors. In this paper, we review the research progress on the mechanism of m6A methylation on the occurrence, development, and tumor immunity of various hematological tumors.

Keywords:  N6-methyladenosine ; hematological tumor ; methylation ; tumor immunity

DOI:  https://doi.org/10.19746/j.cnki.issn.1009-2137.2022.05.048
J Biol Chem. 2022 Sep 28. pii: S0021-9258(22)00992-9. [Epub ahead of print] 102548

Elucidation of the substrate of tRNA-modifying enzymes MnmEG leads to in vitro reconstitution of an evolutionarily conserved uridine hypermodification.

Praneeth Bommisetti, Anthony Young, Vahe Bandarian.

  The evolutionarily conserved bacterial proteins MnmE and MnmG collectively install a carboxymethylaminomethyl (cmnm) group at the fifth position of wobble uridines of several tRNA species. While the reaction catalyzed by MnmEG is one of the central steps in the biosynthesis of the methylaminomethyl (mnm) post-transcriptional tRNA modification, details of the reaction remain elusive. Glycine is known to be the source of the carboxy methylamino moiety of cmnm, and a tetrahydrofolate (THF) analog is thought to supply the one-carbon that is appended to the 5th position of U. However, the nature of the folate analog remains unknown. This manuscript reports the in vitro biochemical reconstitution of the MnmEG reaction. Using isotopically labelled methyl and methylene THF analogs, we demonstrate that methylene THF is the true substrate. We also show that reduced FAD is required for the reaction and that DTT can replace the NADH in its role as a reductant. We discuss the implications of these methylene-THF and reductant requirements on the mechanism of this key tRNA modification catalyzed by MnmEG.

Keywords:  RNA modifications; Transfer RNA; nucleic acid enzymology; tRNA methyltransferase

DOI:  https://doi.org/10.1016/j.jbc.2022.102548
Front Genet. 2022 ;13 984033

Role of 5-methylcytosine in determining the prognosis, tumor microenvironment, and applicability of precision medicine in patients with hepatocellular carcinoma.

Mingyuan Luan, Min Zhao, Haiying Wang, Rongjian Xu, Jinzhen Cai.

  Background: 5-methylcytosine has a profound impact on the development and progression of hepatocellular carcinoma. The aim of this study was to investigate the usefulness of 5-methylcytosine in determining the prognosis, tumor microenvironment, and applicability of precision medicine in hepatocellular carcinoma. Methods: We collected data of seven hepatocellular carcinoma cohorts (The Cancer Genome Atlas, International Cancer Genome Consortium, GSE14520, GSE6764, GSE9843, GSE63898, GSE76427). An unsupervised clustering method was used to identify novel subtypes of hepatocellular carcinoma based on the expression 5-methylcytosine gene signatures. The 5-methylcytosine score was determined using the least absolute shrinkage and selection operator method based on the differential expression of genes in the identified subtypes. Subsequently, we investigated the association between 5-methylcytosine-based clusters (according to the 5-methylcytosine score) and clinical outcomes, immunophenotypes, classical molecular subtypes, and therapeutic opportunities in hepatocellular carcinoma. Finally, we examined the sensitivity of patients with high 5-methylcytosine score to drugs. Results: We identified two hepatocellular carcinoma-specific, 5-methylcytosine-based subtypes (clusters 1 and 2). Cluster 1 exhibited significantly higher 5-methylcytosine scores versus cluster 2. The 5-methylcytosine-based subtypes accurately predicted classical molecular subtypes, immunophenotypes, prognosis, and therapeutic opportunities for patients with hepatocellular carcinoma. Cluster 1 (high 5-methylcytosine score) was characterized by lower anticancer immunity and worse prognosis versus cluster 2 (low 5-methylcytosine score). Moreover, cluster 1 (high 5-methylcytosine score) exhibited low sensitivity to cancer immunotherapy, but high sensitivity to radiotherapy and targeted therapy with lenvatinib. Conclusion: The novel 5-methylcytosine-based subtypes (according to the 5-methylcytosine score) may reflect the prognosis, tumor microenvironment, and applicability of precision medicine in patients with hepatocellular carcinoma.

Keywords:  5 mC methylation; biomarker; hepatocellular carcinoma (HCC); immune escape; immunotherapy

DOI:  https://doi.org/10.3389/fgene.2022.984033
J Transl Med. 2022 Oct 04. 20(1): 450

High expression of HNRNPR in ESCA combined with 18F-FDG PET/CT metabolic parameters are novel biomarkers for preoperative diagnosis of ESCA.

Xiao-Yu Liu, Yan Gao, Xue-Yan Kui, Xu-Sheng Liu, Yao-Hua Zhang, Yu Zhang, Chang-Bin Ke, Zhi-Jun Pei.

   BACKGROUND: The aim of this study was to determine the expression and function of heterogeneous nuclear ribonucleoprotein R (HNRNPR) in esophageal carcinoma (ESCA), the correlation between its expression and 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography/computerized tomography scan (PET/CT)-related parameters. We also investigated whether 18F-FDG PET/CT can be used to predict the expression of HNRNPR in ESCA.
METHODS: We analyzed patients with ESCA who underwent 18F-FDG PET/CT before surgery, and their tissues were stained with HNRNPR IHC. The associated parameters were derived using the 18F-FDG PET imaging data, and the correlation with the IHC score was evaluated. The Oncomine, TCGA, and GEO datasets were used to investigate HNRNPR expression in the pan- and esophageal cancers, as well as its relationship with N6-methyladenosine (m6A) modification and glycolysis. The R software, LinkedOmics, GeneMANIA, and StringOnline tools were used to perform GO/KEGG, GGI, and PPI analyses on the HNRNPR.
RESULTS: HNRNPR is highly expressed in the majority of pan-cancers, including ESCA, and is associated with BMI, weight, and history of reflux in patients with ESCA. HNRNPR is somewhat accurate in predicting the clinical prognosis of ESCA. HNRNPR expression was positively correlated with SUVmax, SUVmean, and TLG in ESCA (p < 0.05). The combination of these three variables provides a strong predictive value for HNRNPR expression in ESCA. GO/KEGG analysis showed that HNRNPR played a role in the regulation of cell cycle, DNA replication, and the Fannie anemia pathway. The analysis of the TCGA and GEO data sets revealed a significant correlation between HNRNPR expression and m6A and glycolysis-related genes. GSEA analysis revealed that HNRNPR was involved in various m6A and glycolysis related-pathways.
CONCLUSION: HNRNPR overexpression correlates with 18F-FDG uptake in ESCA and may be involved in the regulation of the cell cycle, m6A modification, and cell glycolysis. 18F-FDG PET/CT-related parameters can predict the diagnostic accuracy of HNRNPR expression in ESCA.

Keywords:  Esophageal carcinoma (ESCA); Glycolysis; Heterogeneous nuclear ribonucleoprotein R (HNRNPR); PET/CT; m6A modification

DOI:  https://doi.org/10.1186/s12967-022-03665-4
Genomics Proteomics Bioinformatics. 2022 Sep 29. pii: S1672-0229(22)00123-1. [Epub ahead of print]

NSUN2-mediated mRNA m5C Modification Regulates the Progression of Hepatocellular Carcinoma.

Dan Song, Ke An, Wenlong Zhai, Luyao Feng, Yingjie Xu, Ran Sun, Yueqin Wang, Yun-Gui Yang, Quancheng Kan, Xin Tian.

  RNA modification affects many biological processes and physiological diseases. The 5-methylcytosine (m5C) modification regulates the progression of multiple tumors. However, its characteristics and functions in hepatocellular carcinoma (HCC) remain largely unknown. Here, we found that HCC tissues had a higher m5C methylation level than the adjacent normal tissues. Transcriptome analysis revealed that a major function of the hypermethylated genes participated in the phosphokinase signaling pathways, such as the Ras and PI3K-Akt pathways. The m5C methyltransferase NSUN2 was highly expressed in HCC tissues. Interestingly, the expression of many genes was positively correlated with the expression of NSUN2, including GRB2, RNF115, AATF, ADAM15, RTN3, and HDGF. Real-time PCR assays further revealed that the expression of the mRNA of GRB2, RNF115, and AATF decreased significantly with the down-regulation of NSUN2 in HCC cells. Furthermore, NSUN2 could regulate the cellular sensitivity of HCC cells to sorafenib via modulating the Ras signaling pathway. Moreover, knocking down NSUN2 caused cell cycle arrest. Our study demonstrated the vital role of NSUN2 in the progression of HCC.

Keywords:  HCC; NSUN2; Ras pathway; Sorafenib; m(5)C

DOI:  https://doi.org/10.1016/j.gpb.2022.09.007
J Biochem Mol Toxicol. 2022 Oct 07. e23239

Identification of an m6A-related lncRNA prognostic signature in colorectal cancer.

Zhi-Liang Shi, Xiaoling Yang, Cheng-Long Shen, Guo-Qiang Zhou.

  Data sets of colorectal cancer (CRC) were obtained from The Cancer Genome Atlas (TCGA), three N6-methyladenosine (m6A) subtypes were identified using 21 m6A-related long noncoding RNAs (lncRNAs) and differential m6A subtypes of different CRC tumors were determined in this study to evaluate the m6A expression and the prognosis of patients with CRC. Subsequently, eight key lncRNAs were identified based on co-expression with 21 m6A-related genes in CRC tumors using the single-factor Cox and least absolute shrinkage and selection operator. Finally, an m6A-related lncRNA risk score model of CRC tumor was established using multifactor Cox regression based on the eight important lncRNAs and found to have a better performance in evaluating the prognosis of patients in the TCGA-CRC data set. TCGA-CRC tumor samples were divided based on the risk scores: high and low. Then, the clinical characteristics, tumor mutation load, and tumor immune cell infiltration difference between the high- and low-risk-score groups were explored, and the predictive ability of the risk score was assessed for immunotherapeutic benefits. We found that the risk score model can determine the overall survival, be a relatively independent prognostic indicator, and better evaluate the immunotherapeutic benefits for patients with CRC. This study provides data support for accurate immunotherapy in CRC.

Keywords:  M6A; colorectal cancer; lncRNA; prognostic signature

DOI:  https://doi.org/10.1002/jbt.23239
Cancer Res. 2022 Oct 05. pii: CAN-22-0848. [Epub ahead of print]

Loss of LOXL2 promotes uterine hypertrophy and tumor progression by enhancing H3K36ac-dependent gene expression.

Xufeng Lu, Dazhuan E Xin, Juanjuan K Du, Quanli C Zou, Qian Wu, Yanan S Zhang, Wenhai Deng, Jicheng Yue, Xing S Fan, Yuanyuan Zeng, Xiaju Cheng, Xue Li, Zhaoyuan Hou, Man Mohan, Ting C Zhao, Xiaomei Lu, Zhijie Chang, Li-Yan Xu, Yu Sun, Xiongbing Zu, Yu Zhang, Y Eugene Chin.

Lysyl oxidase-like 2 (LOXL2) is a member of the scavenger-receptor cysteine-rich (SRCR) repeat carrying LOX family. Although LOXL2 is suspected to be involved in histone association and chromatin modification, the role of LOXL2 in epigenetic regulation during tumorigenesis and cancer progression remains unclear. Here, we report that nuclear LOXL2 associates with histone H3 and catalyzes H3K36ac deacetylation and deacetylimination. Both the N-terminal SRCR repeats and the C-terminal catalytic domain of LOXL2 carry redundant deacetylase catalytic activity. Overexpression of LOXL2 markedly reduced H3K36 acetylation and blocked H3K36ac-dependent transcription of genes, including c-MYC, CCND1, HIF1A, and CD44. Consequently, LOXL2 overexpression reduced cancer cell proliferation in vitro and inhibited xenograft tumor growth in vivo. In contrast, LOXL2 deficiency resulted in increased H3K36 acetylation and aberrant expression of H3K36ac-dependent genes involved in multiple oncogenic signaling pathways. Female LOXL2 deficient mice spontaneously developed uterine hypertrophy and uterine carcinoma. Moreover, silencing LOXL2 in cancer cells enhanced tumor progression and reduced the efficacy of cisplatin and anti-programmed cell death 1 (PD-1) combination therapy. Clinically, low nuclear LOXL2 expression and high H3K36ac levels corresponded to poor prognosis in uterine endometrial carcinoma patients. These results suggest that nuclear LOXL2 restricts cancer development in the female reproductive system via regulation of H3K36ac deacetylation.

DOI: https://doi.org/10.1158/0008-5472.CAN-22-0848
Front Pharmacol. 2022 ;13 1010879

Nucleophosmin 1 is a prognostic marker of gastrointestinal cancer and is associated with m6A and cuproptosis.

Xu-Sheng Liu, Chao Liu, Jing Zeng, Dao-Bing Zeng, Yi-Jia Chen, Fan Tan, Yan Gao, Xiao-Yu Liu, Yu Zhang, Yao-Hua Zhang, Zhi-Jun Pei.

  Background: NPM1 is highly expressed in a variety of solid tumors and promotes tumor development. However, there are few comprehensive studies on NPM1 analysis in gastrointestinal cancer. Methods: We used bioinformatics tools to study the expression difference of NPM1 between gastrointestinal cancer and control group, and analyzed the relationship between its expression level and the diagnosis, prognosis, functional signaling pathway, immune infiltration, m6A and cuproptosis related genes of gastrointestinal cancer. At the same time, the expression difference of NPM1 between esophageal carcinoma (ESCA) samples and control samples was verified by in vitro experiments. Results: NPM1 was overexpressed in gastrointestinal cancer. In vitro experiments confirmed that the expression of NPM1 in ESCA samples was higher than that in normal samples. The expression of NPM1 has high accuracy in predicting the outcome of gastrointestinal cancer. The expression of NPM1 is closely related to the prognosis of multiple gastrointestinal cancers. Go and KEGG enrichment analysis showed that NPM1 co-expressed genes involved in a variety of biological functions. NPM1 expression is potentially associated with a variety of immune cell infiltration, m6A and cuproptosis related genes in gastrointestinal cancers. Conclusion: NPM1 can be used as a diagnostic and prognostic marker of gastrointestinal cancer, which is related to the immune cell infiltration and the regulation of m6A and cuproptosis.

Keywords:  NPM1; cuproptosis; gastrointestinal cancer; immune infiltrates; m6A

DOI:  https://doi.org/10.3389/fphar.2022.1010879
Front Immunol. 2022 ;13 1014861

m6A methylation regulators as predictors for treatment of advanced urothelial carcinoma with anti-PDL1 agent.

Jianqiu Kong, Sihong Lu, Long Zhang, Yuhui Yao, Jie Zhang, Zefeng Shen, Mingli Luo, Bin Liu, Junjiong Zheng, Tianxin Lin.

   Purpose: Immune checkpoint blockade agents were shown to provide a survival advantage in urothelial carcinoma, while some patients got minimal benefit or side effects. Therefore, we aimed to investigate the prognostic value of m6A methylation regulators, and developed a nomogram for predicting the response to atezolizumab in urothelial carcinoma patients.
Methods: A total of 298 advanced urothelial carcinoma patients with response data in the IMvigor210 cohort were included. Differential expressions of 23 m6A methylation regulators in different treatment outcomes were conducted. Subsequently, a gene signature was developed in the training set using the least absolute shrinkage and selection operator (LASSO) regression. Based on the multivariable logistic regression, a nomogram was constructed by incorporating the gene signature and independent clinicopathological predictors. The performance of the nomogram was assessed by its discrimination, calibration, and clinical utility with internal validation.
Results: Six m6A methylation regulators, including IGF2BP1, IGF2BP3, YTHDF2, HNRNPA2B1, FMR1, and FTO, were significantly differentially expressed between the responders and non-responders. These six regulators were also significantly correlated with the treatment outcomes. Based on the LASSO regression analysis, the gene signature consisting of two selected m6A methylation regulators (FMR1 and HNRNPA2B1) was constructed and showed favorable discrimination. The nomogram integrating the gene signature, TMB, and PD-L1 expression on immune cells, showed favorable calibration and discrimination in the training set (AUC 0.768), which was confirmed in the validation set (AUC 0.755). Decision curve analysis confirmed the potential clinical usefulness of the nomogram.
Conclusions: This study confirmed the prognostic value of FMR1 and HNRNPA2B1, and constructed a nomogram for individualized prediction of the response to atezolizumab in patients with urothelial carcinoma, which may aid in making treatment strategies.

Keywords:  PD1/PDL1; m6A methylation regulators; outcome; prediction; urothelial carcinoma

DOI:  https://doi.org/10.3389/fimmu.2022.1014861
J Cancer Res Clin Oncol. 2022 Oct 07.

N6-methyladenosine long non-coding RNAs reveal novel tool to implicate overall survival and immune microenvironment in renal clear cell carcinoma.

Jingchao Liu, Jinfu Wang, Lanxin Zhang, Jiawen Wang, Zhengtong Lv, Yaoguang Zhang, Jianye Wang.

   PURPOSE: This study aimed to investigate whether N6-methyladenosine (m6A)-related long non-coding RNAs (m6ARelncRNAs) could provide novel tools to predict overall survival of renal clear cell carcinoma.
METHODS: The transcriptomic data and clinical information of patients with renal clear cell carcinoma from The Cancer Genome Atlas (TCGA) were analysed. Distinct m6A modification patterns were systemically analysed via consensus clustering analysis. An m6ARelncRNA signature was constructed in the training cohort using the least absolute shrinkage and selection operator (LASSO) analysis and validated in the test cohort. Potential predictive accuracy of the signature was further assessed via Kaplan-Meier survival, univariate and multivariate Cox regression and subgroup analyses. The Tumour Immune Dysfunction and Exclusion (TIDE) algorithm was used to investigate the role of m6ARelncRNAs in guiding immunotherapy for patients with renal carcinoma.
RESULTS: An m6ARelncRNA signature based on only six lncRNAs was successfully constructed. The high-risk group derived from this signature had significantly poorer overall survival in both training and test cohorts (p < 0.001). Independent prognostic analysis further revealed that m6ARelncRNA risk (p < 0.01) was an independent risk factor for survival outcomes of renal carcinoma. TIDE algorithm revealed that immunotherapy response was poorer in the high-risk group than in the low-risk group. Drug sensitivity analysis based on IC50 revealed that high-risk patients were potentially sensitive to various anti-tumour drugs, including bortezomib, cisplatin, docetaxel, etoposide and sunitinib.
CONCLUSION: m6ARelncRNAs provide novel tools that can be used to predict overall survival and examine the immune microenvironment of renal clear cell carcinoma.

Keywords:  Drug sensitivity analysis; Immune microenvironment; Renal clear cell carcinoma; lncRNAs; m6A

DOI:  https://doi.org/10.1007/s00432-022-04389-0
Cell Death Dis. 2022 Oct 04. 13(10): 848

Upregulation of CRABP2 by TET1-mediated DNA hydroxymethylation attenuates mitochondrial apoptosis and promotes oxaliplatin resistance in gastric cancer.

Xiaolong Tang, Yahang Liang, Guorui Sun, Qingsi He, Zhenyu Hou, Xingzhi Jiang, Peng Gao, Hui Qu.

Oxaliplatin is the main chemotherapy drug for gastric cancer (GC), but quite a few patients are resistant to oxaliplatin, which contributes to the poor prognosis of GC patients. There is therefore an urgent need to identify potential targets for reversing chemotherapy resistance in GC patients. In this study, we analyzed the tumor samples of GC patients who received neoadjuvant chemotherapy based on oxaliplatin through quantitative proteomics and identified the potential chemoresistance-related protein cellular retinoic acid binding protein 2 (CRABP2). CRABP2 was significantly upregulated in the tumor tissues of chemoresistant GC patients and was closely related to prognosis. The results of cell function experiments showed that CRABP2 can promote the oxaliplatin resistance of GC cells in vitro. Coimmunoprecipitation and GST pulldown assays showed that CRAPB2 expedited the binding of BAX and PARKIN in GC cells and facilitated the ubiquitination-mediated degradation of BAX. Furthermore, both the in vitro assay and cell-derived xenograft (CDX) in vivo model verified that CRABP2 promoted oxaliplatin resistance by inhibiting BAX-dependent cell apoptosis. Further experiments proved that the abnormally high expression of CRABP2 in oxaliplatin-resistant GC cells was affected by TET1-mediated DNA hydroxymethylation. The patient-derived xenograft (PDX) model suggested that interference with CRABP2 reversed oxaliplatin resistance in GC in vivo. In conclusion, the results of our study show that CRABP2 was a key molecule in oxaliplatin resistance regulation and could be a new target for reversing the chemoresistance of GC.

DOI: https://doi.org/10.1038/s41419-022-05299-2
Am J Physiol Gastrointest Liver Physiol. 2022 Oct 04.

IMP1/IGF2BP1 in human colorectal cancer extracellular vesicles.

Madeline Kuhn, Yang Zhang, John Favate, Mayu Morita, Aurora Blucher, Sukanya Das, Shun Liang, Ranjan Preet, Louis R Parham, Kathy N Williams, Sudheer Molugu, Randall J Armstrong, Wei Zhang, Jiegang Yang, Kathryn E Hamilton, Dan A Dixon, Gordon Mills, Terry K Morgan, Premal Shah, Sarah F Andres.

  Colorectal cancer (CRC) is a leading cause of cancer-related death. There is an urgent need for new methods of early CRC detection and monitoring to improve patient outcomes. Extracellular vesicles (EVs) are secreted, lipid-bilayer bound, nanoparticles that carry biological cargo throughout the body and in turn exhibit cancer-related biomarker potential. RNA binding proteins (RBPs) are post-transcriptional regulators of gene expression that may provide a link between host cell gene expression and EV phenotypes. Insulin-like growth factor 2 RNA binding protein 1 (IGF2BP1/IMP1) is an RBP that is highly expressed in CRC with higher levels of expression correlating with poor prognosis. IMP1 binds and potently regulates tumor-associated transcripts that may impact CRC EV phenotypes. Our objective was to test whether IMP1 expression levels impact EV secretion and/or cargo. We used RNA sequencing, in vitro CRC cell lines, ex vivo colonoid models, and xenograft mice to test the hypothesis that IMP1 influences EV secretion and/or cargo in human CRC. Our data demonstrate that IMP1 modulates the RNA expression of transcripts associated with extracellular vesicle pathway regulation, but it has no effect on EV secretion levels in vitro or in vivo. Rather, IMP1 appears to affect EV regulation by directly entering EVs in a transformation-dependent manner. These findings suggest that IMP1 has the ability to shape EV cargo in human CRC, which could serve as a diagnostic/prognostic circulating tumor biomarker.

Keywords:  RNA binding protein; colorectal adenocarcinoma; exosome; liquid biopsy

DOI:  https://doi.org/10.1152/ajpgi.00121.2022
ACS Chem Biol. 2022 Oct 03.

Chemical Probe-Based Nanopore Sequencing to Selectively Assess the RNA Modifications.

Soundhar Ramasamy, Vinodh J Sahayasheela, Surbhi Sharma, Zutao Yu, Takuya Hidaka, Li Cai, Vaijayanthi Thangavel, Hiroshi Sugiyama, Ganesh N Pandian.

Nanopore direct RNA sequencing (dRNA-Seq) reads reveal RNA modifications through consistent error profiles specific to a modified nucleobase. However, a null data set is required to identify actual RNA modification-associated errors for distinguishing it from confounding highly intrinsic sequencing errors. Here, we reveal that inosine creates a signature mismatch error in dRNA-Seq reads and obviates the need for a null data set by harnessing the selective reactivity of acrylonitrile for validating the presence of actual inosine modifications. Selective reactivity of acrylonitrile toward inosine altered multiple dRNA-Seq parameters like signal intensity and trace value. We also deduced the stoichiometry of inosine modification through deviation in signal intensity and trace value using this chemical biology approach. Furthermore, we devised Nano ICE-Seq, a protocol to overcome the low coverage issue associated with direct RNA sequencing. Taken together, our chemical probe-based approach may facilitate the knockout-free detection of disease-associated RNA modifications in clinical scenarios.

DOI: https://doi.org/10.1021/acschembio.2c00221
J Orthop Res. 2022 Oct 07.

Comprehensive analysis of m6A methylation modification in chronic spinal cord injury in mice.

Chengjun Li, Jinyun Zhao, Tian Qin, Yuxin Jin, Chunyue Duan, Tianding Wu, Manini Daudi Romani, Yong Cao, Hongbin Lu, Jianzhong Hu.

  Chronic spinal cord injury (CSCI) is a catastrophic disease of the central nervous system (CNS), resulting in partial or complete loss of neurological function. N6-methyladenosine (m6A) is the most common form of reversible posttranslational modification at the RNA level. However, the role of m6A modification in CSCI remains unknown. In this study, we established a CSCI model using a water-absorbable polyurethane polymer, with behavioral assessment, electrophysiological analysis, and histochemical staining for validation. Methylated RNA immunoprecipitation sequencing (meRIP-seq) and mRNA sequencing (mRNA-seq) were jointly explored to compare the differences between CSCI spinal tissue and normal spinal tissue. Furthermore, qRT-PCR, western blotting, and immunofluorescence staining were used to analyze m6A modification-related proteins. We found that water-absorbable polyurethane polymer simulated well chronic spinal cord compression. BMS scores and electrophysiological analysis showed continuous neurological function decline after chronic compression of the spinal cord. meRIP-seq identified 642 differentially modified m6A genes, among which 263 genes were downregulated and 379 genes were upregulated. mRNA-seq showed that 1544 genes were upregulated and 290 genes were downregulated after CSCI. Gene Ontology (GO) terms and enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were also identified. qRT-PCR, western blotting, and immunofluorescence staining showed that Mettl14, Ythdf1 and Ythdf3 were significantly upregulated after CSCI. Our study revealed a comprehensive profile of m6A modifications in CSCI which may act as a valuable key for future research on CSCI. This article is protected by copyright. All rights reserved.

Keywords:  chronic spinal cord injury; m6A; m6A-related protein; mRNA-seq; meRIP sequencing

DOI:  https://doi.org/10.1002/jor.25457