bims-rimeca 2023-01-22 papers

bims-rimeca

Biomed News

on RNA methylation in cancer

Issue of 2023–01–22
33 papers selected by
Sk Ramiz Islam, Saha Institute of Nuclear Physics

N6-methyladenosine writer METTL3 accelerates the sepsis-induced myocardial injury by regulating m6A-dependent ferroptosis.
USP1/UAF1-stabilized METTL3 promotes reactive astrogliosis and improves functional recovery after spinal cord injury through m6A modification of YAP1 mRNA.
METTL3-Mediated N6-Methyladenosine Modification of lncRNA D26496 Suppresses the Proliferation and Migration of Schwann Cells after Sciatic Nerve Injury.
The m6A Reader YTHDF2 Modulates Antiviral and Antibacterial Activity by Suppressing METTL3 Methylation-Modified STING in Fish.
Effect of circular RNAs and N6-methyladenosine (m6A) modification on cancer biology.
Bioinformatics analysis of the correlation between m6A RNA methylation regulators and the immune infiltration and prognosis of bladder cancer.
N6-Methyladenosine Methyltransferase METTL3 Alleviates Diabetes-Induced Testicular Damage through Modulating TUG1/Clusterin Axis.
YTHDF2 orchestrates tumor-associated macrophage reprogramming and controls antitumor immunity through CD8+ T cells.
Identification and Validation of Three m6A Regulators: FTO, HNRNPC, and HNRNPA2B1 as Potential Biomarkers for Endometriosis.
WTAP-mediated m6A modification modulates bone marrow mesenchymal stem cells differentiation potential and osteoporosis.
Suppression of YTHDF2 attenuates autoimmune hepatitis by expansion of myeloid-derived suppressor cells.
The landscape of m6A regulators in esophageal cancer: molecular characteristics, immuno-oncology features, and clinical relevance.
5-Methylcytosine (m5C) Modification Patterns and Tumor Immune Infiltration Characteristics in Clear Cell Renal Cell Carcinoma.
IGFBP3 epigenetic promotion induced by METTL3 boosts cardiac fibroblast activation and fibrosis.
YTHDF1 enhances chondrogenic differentiation by activating the Wnt/β-catenin signaling pathway.
Transcriptome-wide m6A modification mediates cardiotoxicity in mice after chronic exposure to microplastics.
Integrated Profiles of Transcriptome and mRNA m6A Modification Reveal the Intestinal Cytotoxicity of Aflatoxin B1 on HCT116 Cells.
M2-TAMs promote immunoresistance in lung adenocarcinoma by enhancing METTL3-mediated m6A methylation.
Evolution of m6A-related genes in insects and the function of METTL3 in silkworm embryonic development.
YTHDF2 in dentate gyrus is the m6A reader mediating m6A modification in hippocampus-dependent learning and memory.
N6-methyladenosine methylation-related genes YTHDF2, METTL3, and ZC3H13 predict the prognosis of hepatocellular carcinoma patients.
Overexpression of YTHDC2 contributes to the progression of prostate cancer and predicts poor outcomes in patients with prostate cancer.
DNA Methylation and RNA-DNA Hybrids Regulate the Single-Molecule Localization of a DNA Methyltransferase on the Bacterial Nucleoid.
Effect of N6-methyladenosine (m6A) regulator-related immunogenes on the prognosis and immune microenvironment of breast cancer.
Ascorbic acid protects retinal pigment epithelial cells from high glucose by inhibiting the NF-κB signal pathway through MALAT1/IGF2BP3 axis.
m6A reader Igf2bp3 enables germ plasm assembly by m6A-dependent regulation of gene expression in zebrafish.
Prognostic potential of METTL3 expression in patients with gastric cancer.
Molecular subtypes based on cuproptosis-related genes and tumor microenvironment infiltration characteristics in pancreatic adenocarcinoma.
The c-Myc targeting hnRNPAB promotes lung adenocarcinoma cell proliferation via stabilization of CDK4 mRNA.
Association of FTO variant with parental history of type 2 diabetes mellitus in adults.
Foxo3a-Mediated DNMT3B Impedes Cervical Cancer Cell Proliferation and Migration Capacities through Suppressing PTEN Promoter Methylation.
Protocadherin 20 promotes ferroptosis by suppressing the expression of Sirtuin 1 and promoting the acetylation of nuclear factor erythroid 2-related factor 2 in hepatocellular carcinoma.
Epigenetic-Metabolic Interplay in the DNA Damage Response and Therapeutic Resistance of Breast Cancer.

Apoptosis. 2023 Jan 16.

N6-methyladenosine writer METTL3 accelerates the sepsis-induced myocardial injury by regulating m6A-dependent ferroptosis.

Hao Shen, Keliang Xie, Yikui Tian, Xiaoye Wang.

  Ferroptosis is an iron-dependent and phospholipid peroxidation-mediated cell death, which has been identified to be involved in sepsis-induced injury. However, the in-depth molecular mechanisms of N6-methyladenosine (m6A) and ferroptosis on sepsis-induced myocardial injury are still unclear. Here, in the septic myocardial injury, m6A methyltransferase METTL3 level and methylation level high-expressed in lipopolysaccharide (LPS)-induced cardiomyocytes (H9C2). Functionally, METTL3 silencing repressed the ferroptosis phenotype induced by LPS. Mechanistically, METTL3-mediated m6A methylation on solute carrier family 7 member 11 (SLC7A11) empowered its mRNA with high methylation level. Moreover, YTHDF2 directly bound to the m6A modification sites of SLC7A11 to mediate the mRNA degradation. The m6A modified SLC7A11 mRNA was recognized by YTHDF2, which promoted the decay of SLC7A11 mRNA, consequently up-regulating ferroptosis in sepsis-induced myocardial injury. Together, these findings establish a role of METTL3 in the ferroptosis of LPS-induced cardiomyocytes, and provide potential therapeutic target to treat the sepsis-induced myocardial injury.

Keywords:  Ferroptosis; METTL3; Myocardial injury; N6-methyladenosine; Sepsis

DOI:  https://doi.org/10.1007/s10495-022-01808-y
J Neurosci. 2023 Jan 17. pii: JN-RM-1209-22. [Epub ahead of print]

USP1/UAF1-stabilized METTL3 promotes reactive astrogliosis and improves functional recovery after spinal cord injury through m6A modification of YAP1 mRNA.

Xuhui Ge, Wu Ye, Yufeng Zhu, Min Cui, Jiawen Zhou, Chenyu Xiao, Dongdong Jiang, Pengyu Tang, Jiaxing Wang, Zhuanghui Wang, Chengyue Ji, Xuhui Zhou, Xiaojian Cao, Wei Liu, Weihua Cai.

RNA N6-methyladenosine (m6A) modification is involved in diverse biological processes. However, its role in spinal cord injury (SCI) is poorly understood. The m6A level increases in injured spinal cord, and METTL3, which is the core subunit of methyltransferase complex, is upregulated in reactive astrocytes, and further stabilized by the USP1/UAF1 complex after SCI. The USP1/UAF1 complex specifically binds to and subsequently removes K48-linked ubiquitination of the METTL3 protein to maintain its stability after SCI. Moreover, conditional knockout of astrocytic METTL3 in both sexes of mice significantly suppressed reactive astrogliosis after SCI, thus resulting in widespread infiltration of inflammatory cells, aggravated neuronal loss, hampered axonal regeneration, and impaired functional recovery. Mechanistically, the YAP1 transcript was identified as a potential target of METTL3 in astrocytes. METTL3 could selectively methylate the 3'-UTR region of the YAP1 transcript, which subsequently maintains its stability in an IGF2BP2-dependent manner. In vivo, YAP1 overexpression by adeno-associated virus-YAP1 injection remarkably contributed to reactive astrogliosis and partly reversed the detrimental effects of METTL3 knockout on functional recovery after SCI. Furthermore, we found that the methyltransferase activity of METTL3 plays an essential role in reactive astrogliosis and motor repair, whereas METTL3 mutant without methyltransferase function failed to promote functional recovery after SCI. Our study reveals the previously unreported role of METTL3-mediated m6A modification in SCI and might provide a potential therapy for SCI.SIGNIFICANCE STATEMENT:Spinal cord injury is a devastating trauma of the central nervous system involving motor and sensory impairments. However, epigenetic modification in spinal cord injury is still unclear. Here, we propose an m6A regulation effect of astrocytic METTL3 following spinal cord injury and we further characterize its underlying mechanism, which might provide promising strategies for spinal cord injury treatment.

DOI: https://doi.org/10.1523/JNEUROSCI.1209-22.2023
Mol Neurobiol. 2023 Jan 19.

METTL3-Mediated N6-Methyladenosine Modification of lncRNA D26496 Suppresses the Proliferation and Migration of Schwann Cells after Sciatic Nerve Injury.

Xin He, Jia'nan Zhang, Yunshan Guo, Xiaowei Yang, Yunfei Huang, Dingjun Hao.

  Previous reports showed that LncRNA D26496 was downregulated and N6-methyladenosine (m6A) methyltransferase METTL3 was upregulated in sciatic nerve injury (SNI). YTH-Domain Family Member 2 (YTHDF2) regulated RNA degradation through recognizing m6A sites. However, whether METTL3-mediated m6A of D26496 plays a role in development of SNI is unknown. Therefore, in this study, we established a rat SNI model and a H2O2-induced Schwann cell injury model to investigate the role of D26496 in modulating SNI and how the expression of D26496 was regulated during this process. D26496 expression was downregulated in both models. Rats with SNI displayed severe oxidative stress, manifested as increased MDA production and decreased SOD and GSH activity. Moreover, overexpression of D26496 alleviated H2O2-induced Schwann cell injury likely by promoting cell proliferation and migration and suppressing cell apoptosis and oxidative stress. Mechanism studies found that METTL3 expression was upregulated after SNI, and silencing METTL3 reduced the D26496 m6A level, but upregulated D26496 expression. Subsequent studies found that YTHDF2 was upregulated after SNI, and abundant m6A modified D26496 in the precipitated protein-RNA complexes by anti-YTHDF2 antibody, whereas silencing YTHDF2 promoted D26496 expression but had no effect on m6A levels of D29496. Silencing D26496 reversed the protective effect of knocking down METTL3 or knocking down YTHDF2 on H2O2-induced cell damage. In vivo, D26496 overexpression alleviated SNI-induced neuropathic pain and oxidative stress. In conclusion, our results suggested that D26496 m6A modification mediated by METTL3 and recognition of D26496 m6A sites by YTHDF2 induced D26496 degradation, thereby participating in the progression of SNI.

Keywords:  METTL3; Sciatic nerve injury; YTHDF2; lncRNA D26496; m6A

DOI:  https://doi.org/10.1007/s12035-023-03222-0
J Immunol. 2023 Jan 16. pii: ji2200618. [Epub ahead of print]

The m6A Reader YTHDF2 Modulates Antiviral and Antibacterial Activity by Suppressing METTL3 Methylation-Modified STING in Fish.

Shang Geng, Weiwei Zheng, Wansu Wang, Xing Lv, Shiying Xin, Tianjun Xu.

At present, N6-methyladenosine (m6A) modification has been proven to participate in a wide range of gene expression regulation, such as stability, translation, splicing, and output, among others, which has attracted much attention. Unlike mammals, however, the role of m6A in innate immunity of lower invertebrates has not yet been studied. In this study, we found that the total m6A level of Miichthys miiuy increased during Siniperca chuatsi rhabdovirus and Vibrio anguillarum infection, suggesting that m6A may play an important role in the immune process against pathogens in fish. In addition, our study shows that stimulator of IFN genes (STING) plays a dual immune function against viruses and bacteria in fish, and through degrading STING by identifying its m6A methylation site modified by methyltransferase-like 3 (METTL3), YTH domain family 2 (YTHDF2) can weaken the IRF3 and NF-κB-driven signaling pathway, thus weakening the innate immunity and promoting the infection of Siniperca chuatsi rhabdovirus and V. anguillarum to the M. miiuy. Although there have been reports on m6A modification of STING in mammals, it is still unclear whether there is also m6A modification in lower vertebrates, especially in fish. Therefore, our study provides a reference for filling the gap of m6A modification between fish and mammals.

DOI: https://doi.org/10.4049/jimmunol.2200618
Biomed Pharmacother. 2023 Jan 17. pii: S0753-3322(23)00048-3. [Epub ahead of print]159 114260

Effect of circular RNAs and N6-methyladenosine (m6A) modification on cancer biology.

Gong Zhang, Junhui Hou, Chenxue Mei, Xia Wang, Yuan Wang, Kefeng Wang.

  N6-methyladenosine (m6A), as the most abundant and well-known RNA modification, has been found to play an important role in cancer. Circular RNAs (circRNAs) are a class of single-stranded covalently closed RNA molecules generated by the reverse splicing process. Recent studies have revealed the vital roles of circRNAs in many diseases, including tumorigenesis. Accumulating evidence also shows an association between m6A modification and circRNAs. This study aimed to review the interactions between m6A modification and circRNAs and illustrate their roles in tumorigenesis. m6A modification can modulate the biogenesis, translation, cytoplasmic export, degradation, and other functions of circRNAs in different tumors. circRNAs can also modulate m6A modification by affecting writers, erasers, and readers. We focused on the potential regulatory mechanisms and the biological consequences of m6A modification of circRNAs, as well as the interactions in tumors of different systems. Finally, we listed the possible development directions of m6A modification and circRNAs, which might facilitate the clinical application of tumor therapy. AVAILABILITY OF DATA AND MATERIALS: Not applicable. Keywords.

Keywords:  Cancer; Circular RNA; N6-methyladenosine; Therapy; m6A

DOI:  https://doi.org/10.1016/j.biopha.2023.114260
Ann Transl Med. 2022 Dec;10(24): 1386

Bioinformatics analysis of the correlation between m6A RNA methylation regulators and the immune infiltration and prognosis of bladder cancer.

Jiacheng Wu, Xiaolin Wang, Haifei Xu, Jiale Tian, Hao Ji, Jinfeng Zhu, Haifeng Guo, Zhigang Chen.

   Background: To analyze the effect of N6-methyladenosine (m6A) RNA methylation regulators on the immune infiltration and prognosis of bladder cancer (BC). We explored the related signaling pathways and prognosis-related genes to provide candidate targets for the treatment and prognostic evaluation of BC.
Methods: After downloading BC data from The Cancer Genome Atlas (TCGA) database, the expressions of m6A-related genes were obtained. We then performed correlation and sample cluster analysis of the m6A methylation regulator genes as well as difference comparison and survival analysis for the clustered patients using R software. Gene set enrichment analysis (GSEA) was carried out on cluster-grouped samples. Finally, the prognosis-related genes of BC among the m6A methylation regulators were screened.
Results: Genomic alterations in the m6A regulators were linked to a poor BC prognosis. HNRNPA2B1, HNRNPC, IGF2BP2, RBM15, YTHDF1, and YTHDF2 were found to be associated with advanced clinical stages of BC. Furthermore, the current study revealed that the levels of the m6A regulators were related to the expression levels and immune infiltration levels of immune regulators [immunosuppressive factors, immunostimulators, and major histocompatibility complex (MHC) molecules] in BC. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses suggested that in addition to the relevant immune responses, m6A regulators were involved in the poor prognosis of BC via their participation in blood vessels through regulatory RNA binding, telomeric DNA binding, microRNA (miRNA) binding, negative regulation of messenger RNA (mRNA) processing, negative regulation of DNA biosynthesis, branches of morphogenesis, positive regulation of the Notch receptor target gene transcription, etc.
Conclusions: The expression of m6A RNA methylation regulators is closely linked to immune infiltration and prognosis in BC. Thus, it can be utilized as a potential molecular target for the treatment and prognostic assessment of BC.

Keywords:  Bioinformatics; N6-methyladenosine regulating factor (m6A regulating factor); bladder cancer (BC); immune infiltration; prognosis

DOI:  https://doi.org/10.21037/atm-22-5993
Diabetes Metab J. 2023 Jan 19.

N6-Methyladenosine Methyltransferase METTL3 Alleviates Diabetes-Induced Testicular Damage through Modulating TUG1/Clusterin Axis.

Yuan Tian, Yue-Hai Xiao, Chao Sun, Bei Liu, Fa Sun.

   Background: Background: The present study investigated the regulatory effects of N6-methyladenosine (m6A) methyltransferase like-3 (METTL3) in diabetes-induced testicular damage.
Methods: In vivo diabetic mice and high glucose (HG) treated GC-1 spg cells were established. The mRNA and protein expressions were determined by real-time quantitative polymerase chain reaction, Western blot, immunofluorescence and immunohistochemistry staining. Levels of testosterone, blood glucose, cell viability, and apoptosis were detected by enzyme-linked immunosorbent assay, MTT, and flow cytometry, respectively. Molecular interactions were verified by RNA immunoprecipitation and RNA pull-down assay. Histopathological staining was performed to evaluate testicular injury.
Results: METTL3 and long non-coding RNA taurine up-regulated 1 (lncRNA TUG1) were downregulated in testicular tissues of diabetic mice and HG-treated GC-1 spg cells. METTL3 overexpression could reduce the blood glucose level, oxidative stress and testicular damage but enhance testosterone secretion in diabetic mouse model and HG-stimulated GC-1 spg cells. Mechanically, METTL3-mediated m6A methylation enhanced the stability of TUG1, then stabilizing the clusterin mRNA via recruiting serine and arginine rich splicing factor 1. Moreover, inhibition of TUG1/clusterin signaling markedly reversed the protective impacts of METTL3 overexpression on HG-stimulated GC-1 spg cells.
Conclusion: This study demonstrated that METTL3 ameliorated diabetes-induced testicular damage by upregulating the TUG1/clusterin signaling. These data further elucidate the potential regulatory mechanisms of m6A modification on diabetes-induced testicular injury.

Keywords:  Apoptosis; Clusterin; Diabetes mellitus; Methylation; Oxidative stress; RNA, long noncoding

DOI:  https://doi.org/10.4093/dmj.2021.0306
Nat Immunol. 2023 Jan 19.

YTHDF2 orchestrates tumor-associated macrophage reprogramming and controls antitumor immunity through CD8+ T cells.

Shoubao Ma, Baofa Sun, Songqi Duan, Jingjing Han, Tasha Barr, Jianying Zhang, Marc B Bissonnette, Marcin Kortylewski, Chuan He, Jianjun Chen, Michael A Caligiuri, Jianhua Yu.

Despite tumor-associated macrophages (TAMs) playing a key role in shaping the tumor microenvironment (TME), the mechanisms by which TAMs influence the TME and contribute to cancer progression remain unclear. Here, we show that the N6-methyladenosine reader YTHDF2 regulates the antitumor functions of TAMs. YTHDF2 deficiency in TAMs suppressed tumor growth by reprogramming TAMs toward an antitumoral phenotype and increasing their antigen cross-presentation ability, which in turn enhanced CD8+ T cell-mediated antitumor immunity. YTHDF2 deficiency facilitated the reprogramming of TAMs by targeting interferon-γ-STAT1 signaling. The expression of YTHDF2 in TAMs was regulated by interleukin-10-STAT3 signaling. Selectively targeting YTHDF2 in TAMs using a Toll-like receptor 9 agonist-conjugated small interfering RNA reprogrammed TAMs toward an antitumoral phenotype, restrained tumor growth and enhanced the efficacy of PD-L1 antibody therapy. Collectively, our findings describe the role of YTHDF2 in orchestrating TAMs and suggest that YTHDF2 inhibition is an effective approach to enhance cancer immunotherapy.

DOI: https://doi.org/10.1038/s41590-022-01398-6
Genes (Basel). 2022 Dec 28. pii: 86. [Epub ahead of print]14(1):

Identification and Validation of Three m6A Regulators: FTO, HNRNPC, and HNRNPA2B1 as Potential Biomarkers for Endometriosis.

Jiani Sun, Lei Gan, Jing Sun.

   BACKGROUND: N6-methyladenosine is involved in numerous biological processes. However, the significance of m6A regulators in endometriosis is still unclear.
METHODS: We extracted three significant m6A regulators between non-endometriosis and endometriosis patients from GSE6364 and then we used the random forest model to obtain significant m6A regulators. In addition, we used the nomogram model to evaluate the prevalence of endometriosis. The predictive ability of the candidate genes was evaluated through the receiver operating characteristic curves, while the expression of candidate biomarkers was validated via Western blotting. Additionally, according to candidate genes, we identified m6A subtypes based on which functional enrichment analysis and immune infiltration were performed.
RESULTS: Three significant m6A regulators (fat mass and obesity-associated protein, heterogeneous nuclear ribonucleoprotein A2/B1, and heterogeneous nuclear ribonucleoprotein C) were discovered. We identified three m6A subtypes, including clusterA, clusterB, and clusterC. ClusterB was demonstrated to be correlated with significantly overexpressed VEGF and notably downregulated ESR1 and PGR, which are convincing biomarkers of endometriosis. Furthermore, we discovered that patients in clusterB were associated with high levels of neutrophil infiltration, a reduced Treg/Th17 ratio, and overexpressed pyroptosis-related genes, which also indicated that clusterB was highly linked to endometriosis.
CONCLUSION: In conclusion, m6A regulators are of great significance for the occurrence and process of endometriosis. The findings of our study provide novel insights into the underlying molecular mechanism of endometriosis. The novel investigation of m6A patterns and their correlation with immunity may also help to guide the clinical diagnosis, provide prognostic significance, and develop immunotherapy strategies for endometriosis patients.

Keywords:  consensus clustering; endometriosis; immune infiltration; inflammation; m6A RNA methylation regulators; pyroptosis; random forest

DOI:  https://doi.org/10.3390/genes14010086
Cell Death Dis. 2023 Jan 17. 14(1): 33

WTAP-mediated m6A modification modulates bone marrow mesenchymal stem cells differentiation potential and osteoporosis.

Yunhao You, Jincheng Liu, Lu Zhang, Xiang Li, Zhenqian Sun, Zihan Dai, Jinlong Ma, Guangjun Jiao, Yunzhen Chen.

An imbalance in the differentiation potential of bone marrow mesenchymal stem cells (BMSCs) is an important pathogenic mechanism underlying osteoporosis (OP). N6-methyladenosine (m6A) is the most common post-transcriptional modification in eukaryotic cells. The role of the Wilms' tumor 1-associated protein (WTAP), a member of the m6A functional protein family, in regulating BMSCs differentiation remains unknown. We used patient-derived and mouse model-derived samples, qRT-PCR, western blot assays, ALP activity assay, ALP, and Alizarin Red staining to determine the changes in mRNA and protein levels of genes and proteins associated with BMSCs differentiation. Histological analysis and micro-CT were used to evaluate developmental changes in the bone. The results determined that WTAP promoted osteogenic differentiation and inhibited adipogenic differentiation of BMSCs. We used co-immunoprecipitation (co-IP), RNA immunoprecipitation (RIP), methylated RNA immunoprecipitation (MeRIP), RNA pulldown, and dual-luciferase assay to explore the direct mechanism. Mechanistically, the expression of WTAP increased during osteogenic differentiation and significantly promoted pri-miR-181a and pri-miR-181c methylation, which was recognized by YTHDC1, and increased the maturation to miR-181a and miR-181c. MiR-181a and miR-181c inhibited the mRNA expression of SFRP1, promoting the osteogenic differentiation of BMSCs. Our results demonstrated that the WTAP/YTHDC1/miR-181a and miR-181c/SFRP1 axis regulated the differentiation fate of BMSCs, suggesting that it might be a potential therapeutic target for osteoporosis.

DOI: https://doi.org/10.1038/s41419-023-05565-x
J Autoimmun. 2023 Jan 13. pii: S0896-8411(23)00002-1. [Epub ahead of print]135 102993

Suppression of YTHDF2 attenuates autoimmune hepatitis by expansion of myeloid-derived suppressor cells.

Zhuwan Lyu, Bingyuan Huang, Jun Zhang, Qiwei Qian, Xiting Pu, Nana Cui, Yiyan Ou, Bo Li, Zhengrui You, Min Lian, Ruqi Tang, Weihua Chen, Zhicong Zhao, Jiajie Hou, M Eric Gershwin, Haiyan Zhang, Qiang Xia, Xiong Ma.

   BACKGROUND & AIMS: The N6-methyladenosine (m6A) reader YTH domain-containing family protein 2 (YTHDF2) is critically involved in a multiplicity of biological processes by mediating the degradation of m6A modified mRNAs. Based on our current understanding of this process, we hypothesized that YTHDF2 will play a role in the natural history and function of myeloid-derived suppressor cells (MDSC) and in particular in AIH.
APPROACH & RESULTS: We took advantage of YTHDF2 conditional knock-out mice to first address the phenotype and function of MDSCs by flow cytometry. Importantly, the loss of YTHDF2 resulted in a gradual elevation of MDSCs including PMN-MDSCs both in liver and ultimately in the BM. Notably, YTHDF2 deficiency in myeloid cells attenuated concanavalin (ConA)-induced liver injury, with enhanced expansion and chemotaxis to liver. Furthermore, MDSCs from Ythdf2CKO mice had a greater suppressive ability to inhibit the proliferation of T cells. Using multi-omic analysis of m6A RNA immunoprecipitation (RIP) and mRNA sequencing, we noted RXRα as potential target of YTHDF2. Indeed YTHDF2-RIP-qPCR confirmed that YTHDF2 directly binds RXRα mRNA thus promoting degradation and decreasing gene expression. Finally, by IHC and immunofluorescence, YTHDF2 expression was significantly upregulated in the liver of patients with AIH which correlated with the degree of inflammation.
CONCLUSION: Suppression of YTHDF2 enhances the expansion, chemotaxis and suppressive function of MDSCs and our data reveals a unique therapeutical target in immune mediated hepatitis.

Keywords:  Autoimmune hepatitis; Myeloid-derived suppressor cells; N6-methyladenosine; YTHDF2

DOI:  https://doi.org/10.1016/j.jaut.2023.102993
Ann Transl Med. 2022 Dec;10(24): 1347

The landscape of m6A regulators in esophageal cancer: molecular characteristics, immuno-oncology features, and clinical relevance.

Zhe Li, Chunyan Zheng, Liquan Huang, Xiaoyang Yin, Zhongtang Wang, Chengxin Liu, Baosheng Li.

   Background: Squamous cell carcinoma (SCC) and adenocarcinoma (AC) are the two main pathological types of esophageal cancer (EC), which differ in molecular features, genetic variation, and treatment sensitivity. However, as a key process in tumorigenesis and development, the role of N6-methyladenosine (m6A) regulators in esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC) is not fully understood.
Methods: This study systematically compared the role of m6A regulators of ESCC and EAC in terms of molecular characteristics, immuno-oncology characteristics, and clinical relevance, and validated our findings in a long-term follow-up patient cohort.
Results: There were many differences in m6A regulators between ESCC and EAC in terms of expression patterns, genetic variation, association with tumor pathways, immune signatures, and immunotherapy sensitivity. Furthermore, VIRMA was identified as a factor with opposite functional and prognostic effects in ESCC and EAC. ESCC patients with high VIRMA expression and EAC patients with low VIRMA expression had a better prognosis. Single-center data showed that low expression of FTO may be associated with superior immunotherapy efficacy in ESCC patients.
Conclusions: The results herein provide novel ideas for understanding the tumor characteristics, occurrence, and development of ESCC and EAC, and suggest new targets for the treatment and intervention of EC.

Keywords:  N6-methyladenosine (m6A); esophageal adenocarcinoma (EAC); esophageal squamous cell carcinoma (ESCC); immunotherapy; tumor characteristics

DOI:  https://doi.org/10.21037/atm-22-5895
Curr Oncol. 2022 Dec 31. 30(1): 559-574

5-Methylcytosine (m5C) Modification Patterns and Tumor Immune Infiltration Characteristics in Clear Cell Renal Cell Carcinoma.

Can Chen, Lin-Yuan Chen, Jie-Xin Zhang, Hua-Guo Xu.

  Recently, studies have revealed the prognostic value of 5-methylcytosine (m5C) in clear cell renal cell carcinoma (ccRCC). However, the role of m5C methylation in ccRCC immune infiltration and the immunotherapeutic response remains unknown. Based on the mRNA expressions of 14 m5C regulators, we evaluated the m5C modification patterns of 530 tumor samples from the TCGA-ccRCC database. We used the principal component analysis (PCA) algorithm to construct individual patient m5Cscores to facilitate individual analysis of m5C modification patterns in ccRCC patients. We finally defined three different m5C modification patterns. Different clinical features and immune heterogeneity existed among the three patterns, and their immune infiltration characteristics could correspond to different immune phenotypes, including the immune-inflamed, immune-excluded, and immune-desert phenotype. We designed the m5Cscore calculated by the PCA algorithm to measure individual patients' m5C modification patterns. The low m5Cscore group presented with a positive prognosis, increased TMB, and immune activation. Additionally, low m5Cscore patients showed an increased response to immune checkpoint inhibitors. We further the value of the m5Cscore in predicting OS verified in four other tumor cohorts. Our findings revealed that m5C methylation modifications are essential in regulating ccRCC immune infiltration. Assessing single ccRCC patients' m5C modification patterns can fully improve our comprehension of tumor immune characteristics and be used to provide effective personalized immunotherapy strategies for clinical use.

Keywords:  TCGA; ccRCC; immunotherapy; m5C methylation; tumor immune microenvironment

DOI:  https://doi.org/10.3390/curroncol30010044
Eur J Pharmacol. 2023 Jan 16. pii: S0014-2999(23)00005-5. [Epub ahead of print] 175494

IGFBP3 epigenetic promotion induced by METTL3 boosts cardiac fibroblast activation and fibrosis.

Ji-Fei Ding, He Sun, Kai Song, Yang Zhou, Bin Tu, Kai-Hu Shi, Dong Lu, Sheng-Song Xu, Hui Tao.

  Cardiac fibrosis remains an unresolved problem in heart disease. Its etiology is directly caused by the activation and proliferation of cardiac fibroblasts (CFs). However, there is limited information regarding the biological role of cardiac fibroblasts in cardiac fibrosis. Herein, we screened out a gene, IGFBP3, whose expression significantly increased in TGF-β1-stimulated human primary CFs by mining RNA-Seq data for differential and WGCNA analysis. We verified the IGFBP3's expression in TAC surgery, ISO-induced cardiac fibrosis models, and TGFβ1-stimulated mouse primary CFs. We also found that the knockdown of IGFBP3 could inhibit the migration and proliferation ability of CFs. Furthermore, we found that aberrant N6-methyladenosine(m6A) mRNA modifications in the animal model and activated CFs may regulate the expression of IGFBP3 in developing cardiac fibrosis. Silencing METTL3 could downregulate the expression of IGFBP3 and inhibit the activation of CFs and the degree of cardiac fibrosis both in vitro and in vivo. Indeed, we also verified the expression of METTL3 and IGFBP3 in the atrial tissues of patients with AF. Thus, METTL3 may regulate IGFBP3's expression and CFs activation via RNA epigenetic modifications, laying the foundation for a specific and novel therapeutic target in cardiac fibrosis.

Keywords:  Cardiac fibroblasts; IGFBP3; METTL3; Migration; Proliferation; m(6)A

DOI:  https://doi.org/10.1016/j.ejphar.2023.175494
Stem Cells Dev. 2023 Jan 17.

YTHDF1 enhances chondrogenic differentiation by activating the Wnt/β-catenin signaling pathway.

Xiaoming Yang, Youxi Lin, Taiqiu Chen, Wenjun Hu, Pengfei Li, Xuemei Qiu, Bo Yang, Anjing Liang, Wenjie Gao.

Cartilage is derived from the chondrogenic differentiation of stem cells, for which the regulatory mechanism has not been fully elucidated. N6-methyladenosine (m6A) mRNA methylation is the most common posttranscriptional modification in eukaryotic mRNAs and is mediated by m6A regulators. However, whether m6A regulators play roles in chondrogenic differentiation is unknown. Herein, we aim to determine the role of a main m6A reader protein, YTH N6-methyladenosine RNA binding protein 1 (YTHDF1), in chondrogenic differentiation regulation. Western blotting assays found the expression of YTHDF1 increased during chondrogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs). The results of qPCR, western blotting, immunohistochemistry and Alcian blue staining revealed that overexpression of YTHDF1 increased cartilage matrix synthesis and the expression of chondrogenic markers when hBMSCs, ATDC5 cells, or C3H10T1/2 cells were induced to undergo chondrogenesis. Conversely, chondrogenesis was clearly inhibited when YTHDF1 was knocked down in hBMSCs, ATDC5 cells, or C3H10T1/2 cells. Further RNA sequencing and molecular biology experiments found that YTHDF1 activated the Wnt/β-catenin signaling pathway during chondrogenic differentiation. Finally, the effects of overexpression and knockdown of YTHDF1 on chondrogenic differentiation were reversed by inhibiting or activating β-catenin activity. Therefore, we demonstrated that YTDHF1 promoted chondrogenic differentiation through activation of the Wnt/β-catenin signaling pathway.

DOI: https://doi.org/10.1089/scd.2022.0216
Chemosphere. 2023 Jan 14. pii: S0045-6535(23)00144-3. [Epub ahead of print] 137877

Transcriptome-wide m6A modification mediates cardiotoxicity in mice after chronic exposure to microplastics.

Min Zhang, Jun Shi, Huiping Deng.

  The widespread presence of microplastics (MPs) has garnered attention owing to their possible adverse effects. However, the cardiotoxicity of MPs and their underlying mechanism remains unclear. N6-methyladenosine (m6A) modification contributes to post-transcriptional modulation of gene expression, but whether this modification is relevant to MP-induced cardiotoxicity is unknown. First, we detected damage to the myocardial tissue among MP-exposed and -unexposed (control) mice by staining them with hematoxylin and eosin, Masson trichrome, and Oil Red O. Then, we comprehensively measured the transcriptome-wide m6A methylome and m6A-altered genes using high-throughput sequencing assays, such as methylated RNA immunoprecipitation sequencing and RNA sequencing. Our data indicated MP-induced myocardial inflammatory injury and histological alterations in vivo, evidenced by the severity of cardiac fibrosis and increased lipid accumulation. We found 878 increased and 316 decreased methylation peaks mostly distributed in the 3' UTR among the MP-exposed group compared with the control group. We found 779 upregulated and 340 downregulated genes in the MP-exposed group. In addition, conjoint analysis of results from the two high-throughput sequencings showed that 109 and 11 hypermethylated genes were upregulated and downregulated, respectively; 12 and 21 hypomethylated genes were upregulated and downregulated, respectively. Results of the cross-link analysis showed that several potential signals, such as ECM-receptor interactions, cell adhesion molecules, cytokine-cytokine receptor interactions, and NF-κB signaling, contributed to MP-induced cardiotoxicity. Our findings indicated that m6A modifications of genes were involved in MP-induced cardiotoxicity and reported new information regarding the chronic cardiotoxicity caused by MP exposure in mice.

Keywords:  Cardiotoxicity; Methylated RNA immunoprecipitation Sequencing (MeRIP-seq); Microplastics; N6-methyladenosine (m6A); RNA sequencing (RNA-Seq)

DOI:  https://doi.org/10.1016/j.chemosphere.2023.137877
Genes (Basel). 2022 Dec 27. pii: 79. [Epub ahead of print]14(1):

Integrated Profiles of Transcriptome and mRNA m6A Modification Reveal the Intestinal Cytotoxicity of Aflatoxin B1 on HCT116 Cells.

Yajiao Wu, Wenqiang Bao, Jinjin Ren, Chutao Li, Mengting Chen, Dongcheng Zhang, An Zhu.

  Aflatoxin B1 (AFB1) is widely prevalent in foods and animal feeds and is one of the most toxic and carcinogenic aflatoxin subtypes. Existing studies have proved that the intestine is targeted by AFB1, and adverse organic effects have been observed. This study aimed to investigate the relationship between AFB1-induced intestinal toxicity and N6-methyladenosine (m6A) RNA methylation, which involves the post-transcriptional regulation of mRNA expression. The transcriptome-wide m6A methylome and transcriptome profiles in human intestinal cells treated with AFB1 are presented. Methylated RNA immunoprecipitation sequencing and mRNA sequencing were carried out to determine the distinctions in m6A methylation and different genes expressed in AFB1-induced intestinal toxicity. The results showed that there were 2289 overlapping genes of the differentially expressed mRNAs and differentially m6A-methylation-modified mRNAs. After enrichment of the signaling pathways and biological processes, these genes participated in the terms of the cell cycle, endoplasmic reticulum, tight junction, and mitophagy. In conclusion, the study demonstrated that AFB1-induced HCT116 injury was related to the disruptions to the levels of m6A methylation modifications of target genes and the abnormal expression of m6A regulators.

Keywords:  AFB1; cell cycle; endoplasmic reticulum stress; m6A modification; mitophagy; tight junction; transcriptome

DOI:  https://doi.org/10.3390/genes14010079
Ann Transl Med. 2022 Dec;10(24): 1380

M2-TAMs promote immunoresistance in lung adenocarcinoma by enhancing METTL3-mediated m6A methylation.

Lige Wu, Daoan Cheng, Xin Yang, Weiqing Zhao, Cheng Fang, Rui Chen, Mei Ji.

   Background: Immunotherapy has become the first-line treatment for advanced non-small-cell lung cancer (NSCLC), but most patients still fail to benefit or have disease progression following treatment. M2 phenotype tumor-associated macrophages (M2-TAMs) are important cellular components in the immunosuppressive microenvironment of NSCLC, but how they contribute to immunoresistance remains unclear. This study was conducted to investigate the role and mechanism of M2-TAMs in NSCLC immunoresistance.
Methods: We collected postoperative tumor samples for detection of M2-TAMs and other immune cells infiltration by immunofluorescence detection and flow cytometry. We then constructed a non-contact cell co-culture system using Transwell chambers. CCK-8, colony formation, wound healing and invasion assays were performed to evaluated the effect of M2-TAMs on the proliferation, migration and invasion abilities of lung adenocarcinoma (LUAD) cells in vitro. Xenograft model were performed to analysis the effect of M2-TAMs on the tumorigenesis and metastasis of LUAD cells in vivo.
Results: M2-TAMs were greatly increased in the tumor tissue of patients with immunoresistant LUAD. They could significantly promote the proliferation, invasion, and migration of LUAD cells, and improve their resistance to cytotoxic T lymphocytes (CTL) cytotoxicity. Further research showed M2-TAMs could considerably enhance the expression of METTL3 and total m6A RNA level in LUAD cells and interfering with METTL3 could significantly reverse the impairment of M2-TAMs on the efficacy of CTL in killing tumor cells.
Conclusions: In conclusion, M2-TAMs could promote LUAD immunoresistance by enhancing METTL3-mediated m6A methylation. Our results suggest METTL3 could be a potential therapeutic target for reversing immunoresistance and shed new light on the mechanism of M2-TAMs promoting LUAD immunoresistance.

Keywords:  M2 phenotype tumor-associated macrophages (M2-TAMs); METTL3; immunoresistance; non-small cell lung cancer (NSCLC)

DOI:  https://doi.org/10.21037/atm-22-6104
Insect Mol Biol. 2023 Jan 20.

Evolution of m6A-related genes in insects and the function of METTL3 in silkworm embryonic development.

Shuai-Qi Liu, Shun-Ze Jia, Huan Tian, Ying-Hui Li, Kai-Wen Hu, Jian-Guo Tao, Yi-Cheng Lu, Yu-Song Xu, Hua-Bing Wang.

  N6-methyladenosine (m6A) plays a key role in many biological processes. However, the function and evolutionary relationship of m6A-related genes in insects remain largely unknown. Here we analyzed the phylogeny of m6A-related genes among 207 insect species, and found that m6A-related genes are evolutionarily conserved in insects. Subcellular localization experiments of m6A-related protein in BmN cells confirmed that BmYTHDF3 was localized in the cytoplasm, BmMETTL3, BmMETTL14, and BmYTHDC were localized in the nucleus, and FL2D was localized to both the nucleus and cytoplasm. We examined the expression patterns of m6A-related genes during the embryonic development of Bombyx mori. To elucidate the function of BmMETTL3 during the embryonic stage, RNA sequencing was performed to measure changes in gene expression in silkworm eggs after BmMETTL3 knockdown, as well as in BmN cells overexpressing BmMETTL3. The global transcriptional pattern showed that knockdown of BmMETTL3 affected multiple cellular processes, including oxidoreductase activity, transcription regulator activity, and cation binding. In addition, transcriptomic data revealed that many observed DEGs were associated with fundamental metabolic processes including carbon metabolism, purine metabolism, amino acid biosynthesis, and citrate cycle. Interestingly, we found that knockdown of BmMETTL3 significantly affected Wnt and Toll/Imd pathways in embryos. Taken together, these results suggest that BmMETTL3 plays an essential role in the embryonic development of B. mori, and deepen our understanding of the function of m6A-related genes in insects. This article is protected by copyright. All rights reserved.

Keywords:  Embryonic development; Lepidoptera; METTL3; N6 methylation of adenosine; RNA methylation

DOI:  https://doi.org/10.1111/imb.12832
Mol Psychiatry. 2023 Jan 20.

YTHDF2 in dentate gyrus is the m6A reader mediating m6A modification in hippocampus-dependent learning and memory.

Mengru Zhuang, Xiaoqi Geng, Peng Han, Pengfei Che, Fanghao Liang, Chao Liu, Lixin Yang, Jun Yu, Zhuxia Zhang, Wei Dong, Sheng-Jian Ji.

N6-methyladenosine (m6A) has been demonstrated to regulate learning and memory in mice. To investigate the mechanism by which m6A modification exerts its function through its reader proteins in the hippocampus, as well as to unveil the specific subregions of the hippocampus that are crucial for memory formation, we generated dentate gyrus (DG)-, CA3-, and CA1-specific Ythdf1 and Ythdf2 conditional knockout (cKO) mice, respectively. Surprisingly, we found that only the DG-specific Ythdf2 cKO mice displayed impaired memory formation, which is inconsistent with the previous report showing that YTHDF1 was involved in this process. YTHDF2 controls the stability of its target transcripts which encode proteins that regulate the elongation of mossy fibers (MF), the axons of DG granule cells. DG-specific Ythdf2 ablation caused MF overgrowth and impairment of the MF-CA3 excitatory synapse development and transmission in the stratum lucidum. Thus, this study identifies the m6A reader YTHDF2 in dentate gyrus as the only regulator that mediates m6A modification in hippocampus-dependent learning and memory.

DOI: https://doi.org/10.1038/s41380-023-01953-z
Ann Transl Med. 2022 Dec;10(24): 1398

N6-methyladenosine methylation-related genes YTHDF2, METTL3, and ZC3H13 predict the prognosis of hepatocellular carcinoma patients.

Yun Wang, Tianjun Li, Haiping Liu, Yu Liang, Guanqun Wang, Guangming Fu, Mitsuhisa Takatsuki, Haijun Qu, Fanbo Jing, Jing Li, Man Jiang.

   Background: Hepatocellular carcinoma (HCC) is a common primary malignant tumor and cause of cancer-related death in humans. Increasing evidence indicates that an imbalance in N6-methyladenosine (m6A) methylation is strongly linked to the occurrence and development of cancer. We used comprehensive bioinformatics to establish a potential prognostic model of HCC based on m6A methylation-related genes. And case analyses were used to verify the results.
Methods: The clinical data and gene expressions were obtained from The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) databases. The prognostic value of m6A methylation-related genes in HCC patients and their relationship with the immune microenvironment were explored by comprehensive bioinformatics analyses. We also collected pathological specimens from 70 patients with HCC from the Department of Pathology, Affiliated Hospital of Qingdao University, and performed immunohistochemical staining on the specimens. We compared tumor specimens from 27 patients positive for METTL3, YTHDF2, and ZC3H13 staining with their adjacent normal tissues and against 27 patient specimens negative for METTL3, YTHDF2, and ZC3H13. The influence of METTL3, YTHDF2, and ZC3H13 on survival was analyzed, and the prognostic model for METTL3, YTHDF2, and ZC3H13 in HCC was verified by clinical data.
Results: Most m6A methylation-related genes showed significantly different expressions between cancer and normal tissues. Three candidate m6A methylation-related genes (YTHDF2, METTL3, and ZC3H13) were significantly correlated with the overall survival (OS) of HCC patients. A Kaplan-Meier survival analysis indicated a worse prognosis of high-risk patients than that of low-risk patients. Immunological analysis showed that the high-risk group was more likely to have higher follicular helper T cell counts and lower resting memory CD4 T cell counts. The expression of YTHDF2, METTL3, and ZC3H13 was validated by other databases, including the Oncomine database, the Human Protein Atlas (HPA), and the Kaplan-Meier plotter.
Conclusions: Our prognostic model based on m6A methylation-related genes effectively predicted the prognosis of HCC patients.

Keywords:  Hepatocellular carcinoma (HCC); International Cancer Genome Consortium database (ICGC database); N6-methyladenosine methylation-related genes (m6A methylation-related genes); The Cancer Genome Atlas database (TCGA database); prognostic model

DOI:  https://doi.org/10.21037/atm-22-5964
J Biochem Mol Toxicol. 2023 Jan 16. e23308

Overexpression of YTHDC2 contributes to the progression of prostate cancer and predicts poor outcomes in patients with prostate cancer.

Jukun Song, Ganhua You, Xinhai Yin, Guohua Zhu, Wei Wang, Yongwei Yu, Jianguo Zhu.

  YTH domain-containing protein 2 (YTHDC2), a member of N6-methyladenosine (m6A) readers, has been reported to be closely associated with multiple cancer types. However, very little is known about the YTHDC2 gene and its involvement in prostate cancer. YTHDC2 protein expression level was analyzed and correlated to clinical outcomes in prostate cancer patients who underwent prostatectomy in Guizhou Provincial People's Hospital. The YTHDC2 expression level was also detected in prostate cancer cell lines and an immortalized prostate epithelial cell line BPH-1 and RWPE1 by quantitative real-time reverse transcription polymerase chain reaction. Furthermore, we established stable cell lines (DU145 and PC-3) transfected with either empty vector or the full-length YTHDC2 gene and conducted cell function assays in vitro. Fisher's exact test and Pearson χ2 test were employed, Kaplan-Meier method was used for the survival analysis. Of 32 patient samples who enrolled in this study, YTHDC2 was significantly upregulated in prostate cancer (PCa) patients with higher Gleason scores and serum prostate-specific antigen levels. YTHDC2 expression was significantly elevated in all PCa cell lines compared to BPH-1 and RWPE1 (all p < 0.05). Functionally, the enforced expression of YTHDC2 markedly promoted cell growth, migration, and invasion efficacies in prostate cancer cells. Our data indicate that YTHDC2 upregulation may be potentially associated with the prognosis of prostate cancer patients.

Keywords:  TMA; YTHDC2; biomarker; prognostic; prostate cancer

DOI:  https://doi.org/10.1002/jbt.23308
mBio. 2023 Jan 16. e0318522

DNA Methylation and RNA-DNA Hybrids Regulate the Single-Molecule Localization of a DNA Methyltransferase on the Bacterial Nucleoid.

Nicolas L Fernandez, Ziyuan Chen, David E H Fuller, Lieke A van Gijtenbeek, Taylor M Nye, Julie S Biteen, Lyle A Simmons.

  Bacterial DNA methyltransferases (MTases) function in restriction modification systems, cell cycle control, and the regulation of gene expression. DnmA is a recently described DNA MTase that forms N6-methyladenosine at nonpalindromic 5'-GACGAG-3' sites in Bacillus subtilis, yet how DnmA activity is regulated is unknown. To address DnmA regulation, we tested substrate binding in vitro and found that DnmA binds poorly to methylated DNA and to an RNA-DNA hybrid with the DNA recognition sequence. Further, DnmA variants with amino acid substitutions that disrupt cognate sequence recognition or catalysis also bind poorly to DNA. Using superresolution fluorescence microscopy and single-molecule tracking of DnmA-PAmCherry, we characterized the subcellular DnmA diffusion and detected its preferential localization to the replisome region and the nucleoid. Under conditions where the chromosome is highly methylated, upon RNA-DNA hybrid accumulation, or with a DnmA variant with severely limited DNA binding activity, DnmA is excluded from the nucleoid, demonstrating that prior methylation or accumulation of RNA-DNA hybrids regulates the association of DnmA with the chromosome in vivo. Furthermore, despite the high percentage of methylated recognition sites and the proximity to putative endonuclease genes conserved across bacterial species, we find that DnmA fails to protect B. subtilis against phage predation, suggesting that DnmA is functionally an orphan MTase involved in regulating gene expression. Our work explores the regulation of a bacterial DNA MTase and identifies prior methylation and RNA-DNA hybrids as regulators of MTase localization. These MTase regulatory features could be common across biology. IMPORTANCE DNA methyltransferases (MTases) influence gene expression, cell cycle control, and host defense through DNA modification. Predicted MTases are pervasive across bacterial genomes, but the vast majority remain uncharacterized. Here, we show that in the soil microorganism Bacillus subtilis, the DNA MTase dnmA and neighboring genes are remnants of a phage defense system that no longer protects against phage predation. This result suggests that portions of the bacterial methylome may originate from inactive restriction modification systems that have maintained methylation activity. Analysis of DnmA movement in vivo shows that active DnmA localizes in the nucleoid, suggesting that DnmA can search for recognition sequences throughout the nucleoid region with some preference for the replisome. Our results further show that prior DNA methylation and RNA-DNA hybrids regulate DnmA dynamics and nucleoid localization, providing new insight into how DNA methylation is coordinated within the cellular environment.

Keywords:  Bacillus subtilis; epigenetic; replisome; restriction modification; superresolution microscopy

DOI:  https://doi.org/10.1128/mbio.03185-22
Transl Cancer Res. 2022 Dec;11(12): 4303-4314

Effect of N6-methyladenosine (m6A) regulator-related immunogenes on the prognosis and immune microenvironment of breast cancer.

Zhun Yu, Qi He, Guoping Xu.

   Background: Breast cancer is one of the most common malignant tumor and the prognosis remains unsatisfying. Various studies demonstrate that m6A modulators are new predictors of prognosis in immune microenvironment. We aimed to identify several m6A regulator-related immunogenes and explore the relationship between m6A regulator-related immunogenes and breast cancer prognosis as well as the tumor immune microenvironment (TIME).
Methods: RNA sequencing data and clinical information on 21 m6A regulators in 1,047 breast cancer samples were downloaded from The Cancer Genome Atlas (TCGA), and immune gene data were downloaded from InnateDB. Kaplan-Meier survival analysis was conducted with log-rank test using the survival package. An m6A-related immunogene-prognostic signature was then constructed, followed by immune infiltration and checkpoint analyses.
Results: A risk prognostic signature of m6A regulator-related immunogenes, including TOX, PSME2, MCTS1, NFKBIE, SH3BP4, RSPH1, JAK1, MLLT4, and PTGES3, was constructed. Furthermore, univariate and multivariate Cox regression analyses suggested that the tumor stage and risk score could be independent prognostic factors for patients with breast cancer. Immune infiltration analysis showed that the infiltration levels of T cells, memory B cells, activated NK cells, and macrophages between the high- and low-risk groups were significantly different. In addition, checkpoint analyses demonstrated that the levels of immune checkpoint genes, such as those of LAG3, PDCD1, CTLA4, and HAVCR2, were downregulated in the high-risk group compared to those in the low-risk group.
Conclusions: Our findings suggest that the m6A regulator-related risk prognostic signature can predict the prognosis of breast cancer and that it is related to the immune microenvironment.

Keywords:  N6-methyladenosine (m6A); breast cancer; checkpoints; prognosis; tumor immune microenvironment (TIME)

DOI:  https://doi.org/10.21037/tcr-22-1335
Diabet Med. 2023 Jan 20. e15050

Ascorbic acid protects retinal pigment epithelial cells from high glucose by inhibiting the NF-κB signal pathway through MALAT1/IGF2BP3 axis.

Yanming Tian, Wubo Cheng, Haiyan Wang, Chengcheng Zeng, Xueyi Chen.

   BACKGROUND: Diabetic retinopathy (DR) is a common complication of diabetes with nocuous effects on patients' eye health, typically accompanies by excessive inflammation and oxidative stress. Insulin-like growth factor-2 messenger RNA-binding protein 3 (IGF2BP3) was engaged with inflammation, whereas its precise role in the DR process was unclear. And enhanced lncRNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) and decreased ascorbic acid (AA) were also found in DR. This study was to explore the regulatory role and mechanism of IGF2BP3, MALAT1, and AA in the high glucose (HG)-induced retinal pigment epithelial (RPE) cell injury.
METHODS: ARPE-19 cells were treated with HG to establish the in vitro RPE cell injury model. The mRNA and protein levels of the gene were evaluated by qRT-PCR or Western blot. Immunofluorescence detected the translocation condition of the p65 protein. Inflammatory factor levels were detected by ELISA assays. Apoptosis was detected by flow cytometry. The binding interaction of IGF2BP3 and MALAT1 was validated by RIP-qPCR assays.
RESULTS: In high glucose induced RPE cell injury, IGF2BP3 expression, inflammatory response and apoptosis were enhanced. Next, the IGF2BP3 activated the NF-κB signaling to promote the RPE cell injury development. MALAT1 could directly bind with IGF2BP3 and upregulate its expression. In addition, AA ameliorated the HG-induced RPE cell injury through the regulation of MALAT1.
CONCLUSION: Ascorbic acid ameliorated HG-induced RPE cell injury by repressing the NF-κB signaling pathway via modulating the MALAT1/IGF2BP3 axis.

Keywords:  Diabetic retinopathy; IGF2BP3; MALAT1; NF-κB signaling; ascorbic acid

DOI:  https://doi.org/10.1111/dme.15050
Sci Bull (Beijing). 2021 Jun 15. pii: S2095-9273(21)00116-X. [Epub ahead of print]66(11): 1119-1128

m6A reader Igf2bp3 enables germ plasm assembly by m6A-dependent regulation of gene expression in zebrafish.

Fan Ren, Ran Miao, Rui Xiao, Jie Mei.

  Bucky ball (Buc) is involved in germ plasm (GP) assembly during early zebrafish development by regulating GP mRNA expression via an unknown mechanism. The present study demonstrates that an m6A reader Igf2bp3 interacts and colocalizes with Buc in the GP. Similar to the loss of Buc, the genetic deletion of maternal igf2bp3 in zebrafish leads to abnormal GP assembly and insufficient germ cell specification, which can be partially restored by the injection of igf2bp3 mRNA. Igf2bp3 binds to m6A-modified GP-organizer and GP mRNAs in an m6A-dependent manner and prevents their degradation. These findings indicate that the functions of Igf2bp3, a direct effector protein of Buc, in GP mRNA expression and GP assembly involve m6A-dependent regulation; these results emphasize a critical role of m6A modification in the process of GP assembly.

Keywords:  Bucky ball; Germ cell specification; Germ plasm assembly; Igf2bp3; m(6)A reader; m(6)A-dependent regulation

DOI:  https://doi.org/10.1016/j.scib.2021.02.003
Oncol Lett. 2023 Feb;25(2): 64

Prognostic potential of METTL3 expression in patients with gastric cancer.

Yoshinaga Okugawa, Yuji Toiyama, Chengzeng Yin, Ma Ruiya, Akul Goel, Takashi Ichikawa, Hiroki Imaoka, Takahito Kitajima, Tadanobu Shimura, Mikio Kawamura, Hiromi Yasuda, Hiroyuki Fujikawa, Takeshi Yokoe, Ikuyo Mochiki, Masaki Ohi, Kaname Nakatani.

  Methyltransferase-like 3 (METTL3) is a crucial component of the m6A methyltransferase complex, which serves pivotal roles in tumor progression. The present study investigated the prognostic significance of METTL3 expression in gastric cancer (GC). The expression levels of METTL3 were assessed by immunohistochemistry in formalin-fixed paraffin-embedded (FFPE) tissue specimens from 158 patients with GC. Propensity score matching (PSM) analysis was performed to clarify its prognostic potential. METTL3 gene expression was also investigated in fresh frozen specimens from another independent cohort of 57 patients with GC to establish its clinical relevance. Knockdown of METTL3 by small interfering RNA transfection was performed to evaluate its function in vitro. METTL3 expression was significantly higher in cancerous tissues compared with in corresponding normal mucosa (P<0.0001), and high METTL3 expression was an independent prognostic factor for overall and disease-free survival in the FFPE cohort of patients with GC. PSM analysis revealed that elevated METTL3 expression was significantly associated with poor survival outcomes, which was subsequently validated in another cohort of fresh frozen specimens. Knockdown of METTL3 inhibited proliferation, invasion, migration and anoikis resistance in GC cells. In conclusion, METTL3 expression may be used as a clinically feasible prognostic marker and could serve as a potential therapeutic target in patients with GC.

Keywords:  METTL3; RNA methyltransferases; gastric cancer; prognosis

DOI:  https://doi.org/10.3892/ol.2022.13651
Cancer Cell Int. 2023 Jan 16. 23(1): 7

Molecular subtypes based on cuproptosis-related genes and tumor microenvironment infiltration characteristics in pancreatic adenocarcinoma.

Jian Li, Jingyang Yin, Wenhua Li, Huaizhi Wang, Bing Ni.

   BACKGROUND: Multiple molecular subtypes with distinct clinical outcomes in pancreatic adenocarcinoma (PAAD) have been identified in recent years. Cuproptosis is a new form of cell death that likely involved in tumor progression. However, the cuproptosis-related molecular subtypes as well as its mediated tumor microenvironment (TME) cell infiltration characteristics largely remain unclear.
METHODS: Expression profiles of 10 cuproptosis-related genes (CRGs) and their association with patient survival, TME, cancer stemness and drug resistance were studied in 33 cancer types using the TCGA pan-cancer data. Using 437 PAAD samples from five cohorts (TCGA-PAAD cohort and four GEO cohorts), we explored the molecular subtypes mediated by CRGs, along with the associated TME cell infiltration. Unsupervised methods were utilized to perform cuproptosis subtype clustering. The cuproptosis score was constructed using the COX regression model with least absolute shrinkage and selection operator regression (LASSO) algorithm to quantify the cuproptosis characteristics of a single tumor.
RESULTS: The expression of 10 CRGs varies in different cancer types with striking inter- and intra- cancer heterogeneity. We integrated the genomic profiling of the CRGs and identified three distinct cuproptosis subtypes, and found that multi-layer CRG alterations were correlated with patient prognosis and TME cell infiltration characteristics. In addition, a cuproptosis score signature was constructed to predict prognosis, and its clinical impacts were characterized in the TCGA-PAAD cohort. The cuproptosis signature was significantly associated with prognosis, tumor subtypes, CD8 T-cell infiltration, response to immune checkpoint inhibitors (ICIs) and chemotherapeutic drug sensitivity. Furthermore, the expression patterns of CRGs in pancreatic cancer cells and normal controls were validated, which was almost consistent with the results from the public database. The expression level and prognostic predictive capability of DLAT were verified in 97 PAAD patients from our patient cohort.
CONCLUSIONS: These findings may help understand the roles of CRGs in PAAD and the molecular characterization of cuproptosis subtypes. In addition, the cuproptosis score could serve as a promising biomarker for predicting prognosis and response to immunotherapy in PAAD patients.

Keywords:  Cuproptosis; Immunotherapy; Molecular subtypes; Pancreatic adenocarcinoma; Tumor microenvironment

DOI:  https://doi.org/10.1186/s12935-022-02836-z
Int J Biochem Cell Biol. 2023 Jan 16. pii: S1357-2725(23)00011-0. [Epub ahead of print] 106372

The c-Myc targeting hnRNPAB promotes lung adenocarcinoma cell proliferation via stabilization of CDK4 mRNA.

Chen Xu, Bingyan Li, Ning Yu, Bo Yao, Fang Wang, Yide Mei.

  The c-Myc oncoprotein plays a pivotal role in tumorigenesis. The deregulated expression of c-Myc has been linked to a variety of human cancers including lung adenocarcinoma. The oncogenic function of c-Myc has been largely attributed to its intrinsic nature as a transcription factor. Here we reported the RNA binding protein hnRNPAB as a direct transcriptional target of c-Myc by performing quantitative real-time polymerase chain reaction (qRT-PCR), western blot, chromatin immunoprecipitation (ChIP), and luciferase reporter analyses. Flow cytometry, colony formation, and RNA immunoprecipitation (RIP) assays were used to investigate the role of hnRNPAB in lung adenocarcinoma cell proliferation, as well as the underlying mechanism. HnRNPAB was functionally shown to promote lung adenocarcinoma cell proliferation by accelerating G1/S cell cycle progression. Mechanistically, hnRNPAB interacted with and stabilized CDK4 mRNA, thereby increasing CDK4 expression. Moreover, hnRNPAB was able to promote G1/S cell cycle progression and cell proliferation via the regulation of CDK4. HnRNPAB was also revealed as a mediator of the promoting effect of c-Myc on cell proliferation. Together, these findings demonstrate that hnRNPAB is an important regulator of lung adenocarcinoma cell proliferation. They also add new insights into the mechanisms of how c-Myc promotes tumorigenesis.

Keywords:  CDK4; c-Myc; cell cycle progression; hnRNPAB; lung adenocarcinoma

DOI:  https://doi.org/10.1016/j.biocel.2023.106372
J Pak Med Assoc. 2022 Oct;72(10): 2009-2013

Association of FTO variant with parental history of type 2 diabetes mellitus in adults.

Zil-e-Rubab

   OBJECTIVE: To find out the association between fat mass and obesity-associated gene polymorphism and risk factors frequently associated with type 2 diabetes mellitus.
METHODS: The case-control study was conducted from January 2020 to March 2021 at the Ziauddin University, Karachi, and comprised deoxyribonucleic acid samples for fat mass and obesity-associated gene polymorphism from non-diabetic Pakistani population. Group A comprised non-diabetics with parental history of type 2 diabetes mellitus and Group B had controls without parental history of type 2 diabetes mellitus. Analysis was based on restriction fragment length polymorphism and polymerase chain reaction. Data was analysed using SPSS 25.
RESULTS: Of the 150 subjects, 75(50%) each were in Group A and Group B. There were 40 (53.3%) males and 35 (46.7%) females in Group A compared to 35 (46.7%) males and 40(53.3%) females in Group B. Overall, 48% subjects were single and 52 % were married. A difference in frequency of fat mass and obesity-associated gene (rs9939609) alleles, such as TT, AA TA, was noted between the groups (p>0.999). TA allele was found to be associated with Group A (33) 44% (p=0.40), while TT allele was associated with Group B (41) 54% (p=0.414). AA allele was equally distributed between the groups (6) 8% (p=1.00).
CONCLUSIONS: The TT allele of fat mass and obesity-associated gene was found to be an independent allele associated with the risk of developing type 2 diabetes mellitus.

Keywords:   Type 2 diabetes mellitus, FTO gene, BMI, SNP, Multifactorial inheritance, Family history, Genetic variant PCR and RFLP.

DOI:  https://doi.org/10.47391/JPMA.4299
J Invest Surg. 2023 Dec;36(1): 2162170

Foxo3a-Mediated DNMT3B Impedes Cervical Cancer Cell Proliferation and Migration Capacities through Suppressing PTEN Promoter Methylation.

Hongying Li, Yuqin Yuan, Hong Dong, Tinghui Wang, Dunlan Zhang, Limin Zhou, Lu Chen, Xueyan He.

   OBJECTIVE: Cervical cancer is linked with the constitutive activation of growth factors and gene mutations-induced pro-survival signaling pathways. Herein, we purposed to explore the possible molecular mechanism of Foxo3a-mediated DNMT3B in the proliferation and migration of cervical cancer cells via mediating the PTEN promoter methylation.
METHODS: Foxo3a expression in cervical cancer was tested by qRT-PCR and western blot experiments. The cervical cancer cell biological functions with overexpression of Foxo3a were evaluated by CCK-8 assay, Transwell experiment, and flow cytometry, respectively. MS-PCR was utilized for testing the PTEN methylation levels, and ChIP experiment was implemented for evaluating the enrichment of DNMT3B in the PTEN promoter region and the binding of Foxo3a and DNMT3B. The PTEN methylation and interference with Foxo3a expression were performed in cervical cancer cells, and then their impacts on cervical cancer cell biological functions were observed.
RESULTS: FOXO3a was expressed at a low level in cervical cancer, and its overexpression contributed to a reduction in cell proliferative, migratory and invasive capabilities, and an elevation in apoptosis rate. Foxo3a blocked its methylation with the PTEN promoter by repressing DNMT3B activity. Upon treatment with methyltransferase inhibitor (5-aza-dc), the malignant phenotypes of cervical cancer cells were diminished. 5-aza-dc neutralized the impacts of silencing Foxo3a on malignant phenotypes.
CONCLUSION: This research underlines that Foxo3a blocks its methylation with the PTEN promoter by inhibiting DNMT3B activity, which subsequently impedes cervical cancer cell progression.

Keywords:  Cervical cancer; DNMT3B; Foxo3a; PTEN; promoter

DOI:  https://doi.org/10.1080/08941939.2022.2162170
Int J Biochem Cell Biol. 2023 Jan 11. pii: S1357-2725(23)00002-X. [Epub ahead of print]156 106363

Protocadherin 20 promotes ferroptosis by suppressing the expression of Sirtuin 1 and promoting the acetylation of nuclear factor erythroid 2-related factor 2 in hepatocellular carcinoma.

Lv Jun, Wang Chen, Liang Han, Liu Yanmin, Zeng Qinglei, Zhu Pengfei.

  Hepatocellular carcinoma (HCC) is characterized by a poor prognosis. Our previous work suggested that Protocadherin 20 (PCDH20) promoted ferroptosis in HCC. Nevertheless, the underlying mechanism remains elusive. Recently, we found that both the mRNA and protein levels of PCDH20 were upregulated in erastin- or sorafenib-treated HCC cells. Meanwhile, data showed that Sirtuin 1 (SIRT1) was markedly downregulated in PCDH20-SNU-449 cells. Additionally, overexpression of PCDH20 or erastin-treated cells dramatically decreased cell viability and colony-forming capacity of HCC cells, whereas blocking PCDH20 reversed these effects. Moreover, PCDH20 overexpression or treatment with erastin significantly downregulated the expression of SIRT1, Solute carrier family 7 member 11 (SLC7A11), as well as the ferroptosis-related protein glutathione peroxidase 4 (GPX4) and glutathione (GSH), while elevated malondialdehyde (MDA), 2'- 7'-dichlorofluorescein (DCF) and intercellular iron levels. Conversely, knockdown of PCDH20 upregulated SIRT1 and SLC7A11. Immunoprecipitation assay demonstrated that PCDH20 or erastin increased the amount of acetylated nuclear factor erythroid 2-related factor-2 (NRF2). This reducing effect of NRF2 deacetylation by PCDH20 was counteracted by restoring the expression of SIRT1. In addition, PCDH20 lowered the levels of GPX4, GSH, and cell viability, as well as resulted in an elevation in intercellular iron level, MDA, and DCF. These effects were reversed by SIRT1 expression. Besides, PCDH20 could promote ferroptosis by inhibiting SIRT1 from deacetylating NRF2, which led to the downregulation of SLC7A11, GPX4, and GSH both in vivo and in vitro. Our results signals that PCDH20 promotes ferroptosis by suppressing the expression of SIRT1 and thus, promoting the acetylation of NRF2in HCC.

Keywords:  Ferroptosis; Hepatocellular carcinoma; NRF2; PCDH20; SIRT1

DOI:  https://doi.org/10.1016/j.biocel.2023.106363
Cancer Res. 2023 Jan 18. pii: CAN-22-3015. [Epub ahead of print]

Epigenetic-Metabolic Interplay in the DNA Damage Response and Therapeutic Resistance of Breast Cancer.

Chandrima Das, Swagata Adhikari, Apoorva Bhattacharya, Sanjukta Chakraborty, Payel Mondal, Shalini S Yadav, Santanu Adhikary, Clayton Hunt, Kamlesh K Yadav, Shruti Pandita, Siddhartha Roy, John A Tainer, Zamal Ahmed, Tej K Pandita.

Therapy resistance is imposing a daunting challenge on effective clinical management of breast cancer. Although the development of resistance to drugs is multifaceted, reprogramming of energy metabolism pathways is emerging as a central but heterogenous regulator of this therapeutic challenge. Metabolic heterogeneity in cancer cells is intricately associated with alterations of different signaling networks and activation of DNA damage response (DDR) pathways. Here we consider how the dynamic metabolic milieu of cancer cells regulates their DNA damage repair ability to ultimately contribute to development of therapy resistance. Diverse epigenetic regulators are crucial in remodeling the metabolic landscape of cancer. This epigenetic-metabolic interplay profoundly affects genomic stability of the cancer cells as well as their resistance to genotoxic therapies. These observations identify defining mechanisms of cancer epigenetics-metabolism-DNA repair axis that can be critical for devising novel, targeted therapeutic approaches which could sensitize cancer cells to conventional treatment strategies.

DOI: https://doi.org/10.1158/0008-5472.CAN-22-3015