bims-rimeca Biomed News
on RNA methylation in cancer
Issue of 2023‒09‒03
thirty-one papers selected by
Sk Ramiz Islam, Saha Institute of Nuclear Physics

  1. Mol Cancer Res. 2023 Aug 30.
      The pathological significance of the circular RNA DDIT4 (CircDDIT4), which is formed by back-splicing at the 3'-untranslated region (UTR) with a 5' splice acceptor site in exon 2 of linear DDIT4 mRNA, has yet to be determined. Our study found that circDDIT4 is downregulated in prostate cancer (PCa) and functions as a tumor suppressor during PCa progression. By competitively binding to ELAV-like RNA binding protein 1 (ELAVL1/HuR) through its 3'-UTR, circDDIT4 acts as a protein sponge to decrease the expression of PCa-overexpressed anoctamin 7 (ANO7). This promotes PCa cell apoptosis while inhibiting cell proliferation and metastasis. Furthermore, we discovered that N6-methyladenosine (m6A) modification facilitates the biogenesis of circDDIT4. The methyltransferase complex consisting of WTAP/METTL3/METTL14 increases the level of circDDIT4, while the RNA demethylase FTO decreases it. Implications: These findings suggest that abnormal co-transcriptional modification of m6A promotes PCa initiation and progression via a circular RNA-protein-cell signaling network.
  2. Cell Death Dis. 2023 Sep 01. 14(9): 581
      Insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3), an RNA-binding protein, is associated with tumorigenesis and progression. However, the exact molecular mechanisms of IGF2BP3 in colorectal cancer (CRC) oncogenesis, progression, and drug resistance remain unclear. This study found that IGF2BP3 was upregulated in CRC tissues. Clinically, the elevated IGF2BP3 level is predictive of a poor prognosis. Functionally, IGF2BP3 enhances CRC tumorigenesis and progression both in vitro and in vivo. Mechanistically, IGF2BP3 promotes epidermal growth factor receptor (EGFR) mRNA stability and translation and further activates the EGFR pathway by serving as a reader in an N6-methyladenosine (m6A)-dependent manner by cooperating with METTL14. Furthermore, IGF2BP3 increases the drug resistance of CRC cells to the EGFR-targeted antibody cetuximab. Taken together, our results demonstrated that IGF2BP3 was a functional and clinical oncogene of CRC. Targeting IGF2BP3 and m6A modification may therefore offer rational therapeutic targets for patients with CRC.
  3. Chin J Physiol. 2023 Jul-Aug;66(4):66(4): 248-256
      Aberrant glycolytic reprogramming is involved in lung cancer progression by promoting the proliferation of non-small cell lung cancer cells. Paeonol, as a traditional Chinese medicine, plays a critical role in multiple cancer cell proliferation and inflammation. Acyl-CoA dehydrogenase (ACADM) is involved in the development of metabolic diseases. N6-methyladenosine (m6A) modification is important for the regulation of messenger RNA stability, splicing, and translation. Here, we investigated whether paeonol regulates the proliferation and glycolytic reprogramming via ACADM with m6A modification in A549 cells (human non-small cell lung cancer cells). Cell counting kit 8, 5-Bromo-2-deoxyuridine, 5-ethynyl-2'-deoxyuridine (EdU) incorporation, flow cytometry analysis, western blotting and seahorse XFe24 extracellular flux analyzer assays showed that paeonol had a significant inhibitory effect against A549 cell proliferation and glycolysis. Mechanistically, ACADM was a functional target of paeonol. We also showed that the m6A reader YTH domain containing 1 plays an important role in m6A-modified ACADM expression, which is negatively regulated by paeonol, and is involved in A549 cell proliferation and glycolytic reprogramming. These results indicated the central function of paeonol in regulating A549 cell glycolytic reprogramming and proliferation via m6A modification of ACADM.
    Keywords:  Acyl-CoA dehydrogenase; N6-methyladenosine; glycolytic reprogramming; paeonol; proliferation
  4. Cell Biol Int. 2023 Sep 01.
      Transcription factors (TFs) and N6-methyladenosine (m6A) modifiers are involved in tumor progression through transcriptional regulation and posttranscriptional regulation of genes, respectively. However, the crosstalk and role of these two types of gene expression regulators in head and neck squamous cell carcinoma (HNSC) remains poorly understood. In this study, we demonstrate that the TF homeobox-D1 (HOXD1) and the m6A demethylase fat mass and obesity-associated protein (FTO) form a positive feedback loop to promote cell proliferation and survival in HNSC. Clinically, HOXD1 expression is dysregulated in multiple cancer types and is associated with worse prognosis in patients with HNSC, stomach adenocarcinoma, uterine corpus endometrial carcinoma, and pheochromocytoma and paraganglioma. Mechanistically, FTO is overexpressed in HNSC tumor samples and positively regulates HOXD1 expression in an m6A-dependent manner. Functionally, deficiency of HOXD1 relieved the resistance of HNSC cells to apoptosis and arrested tumor cells at the G0/G1 phase, thereby inhibiting cell growth, whereas overexpression of HOXD1 caused the opposite effect. Furthermore, HOXD1 activates the transcription of the oncogenic factor FTO by directly targeting its promoter. Downregulation of FTO mimicked the biological effect of HOXD1 knockdown on HNSC. Importantly, overexpression of HOXD1 significantly rescued the proliferation inhibition and apoptosis promotion of HNSC cells induced by deficiency of FTO. Together, our findings reveal HOXD1 as a novel prognostic predictor and a potential target for HNSC, providing mechanistic insights into the role of the HOXD1-FTO circuit in this cancer.
    Keywords:  FTO; HOXD1; N6-methyladenosine; head and neck squamous cell carcinoma; transcription factor
  5. Transl Oncol. 2023 Aug 27. pii: S1936-5233(23)00150-X. [Epub ahead of print]37 101764
      INTRODUCTION: N6-methyladenosine (m6A) is an emerging epigenetic modification, which plays a crucial role in the development of cancer. Nevertheless, the underlying mechanism of m6A-associated proteins and m6A modification in gallbladder cancer remains largely unknown.MATERIALS AND METHODS: The Gene Expression Omnibus database and tissue microarray were used to identify the key m6A-related gene in gallbladder cancer. The function and mechanism of IGF2BP3 were further investigated by knockdown and overexpression techniques in vitro and in vivo.
    RESULTS: We found that IGF2BP3 was elevated and correlated with poor prognosis in gallbladder cancer, which can be used as an independent prognostic factor for gallbladder cancer. IGF2BP3 accelerated the proliferation, invasion and migration of gallbladder cancer cells in vitro and in vivo. Mechanistically, IGF2BP3 interacted with and augmented the stability of CLDN4 mRNA by m6A modification. Enhancement of CLDN4 reversed the inhibitory effect of IGF2BP3 deficiency on gallbladder cancer. Furthermore, we demonstrated that IGF2BP3 promotes the activation of NF-κB signaling pathway by up-regulation of CLDN4. Overexpression of IGF2BP3 in gallbladder cancer cells obviously promoted the polarization of immunosuppressive phenotype in macrophages. Besides, Gallbladder cancer cells-derived IGF2BP3 up-regulated the levels of STAT3 in M2 macrophages, and promoted M2 polarization.
    CONCLUSIONS: We manifested IGF2BP3 promotes the aggressive phenotype of gallbladder cancer by stabilizing CLDN4 mRNA in an m6A-dependent manner and induces macrophage immunosuppressive polarization, which might offer a new theoretical basis for against gallbladder cancer.
    Keywords:  Claudin-4; Gallbladder cancer; M2 macrophages; N6-methyladenosine; Prognosis
  6. Aging (Albany NY). 2023 Aug 29. 15
      BACKGROUND: The immune checkpoint inhibitors (ICIs) has dramatically changed the therapeutic area of cancers. A great number of patients with CRC exhibit poor response rate to ICI treatment. N6-methyl adenosine (m6A) is closely correlated with the initiation and progression of cancers. To explore the role of methyltransferase-like 16 (METTL16) in CRC treatment.METHODS: Clinical samples and different CRC cell lines were collected. The expression of METTL16 and PD-L1 was determined by qPCR, IHC. Ectopic expression of METTL16 was performed in CRC cells. A co-culture system was established using CRC cells and T cells to measure the immune evasion. Cell viability, apoptosis, migration, and invasion were examined by CCK-8, colony formation, flow cytometry, Transwell, and wound healing assay, respectively. The N6-methyl adenosine (m6A) modification of PD-L1 by METTL16 was investigated by methylated RIP (MeRIP) and RNA stability experiment. In vivo xenograft model was established to measure the effects of METTL16 on CRC growth.
    RESULTS: METTL16 was decreased and PD-L1 was increased in CRC tissues and cell lines. METTL16 enhanced cell proliferation, migration, and invasion, and promoted CRC tumor growth in vivo. METTL16 induced m6A modification and decreased the stability of METTL16 RNA, leading to the suppression of METTL16 level. METTL16 overexpression in CRC cells induced decreased portion of PD-1 positive T cells. Overexpression of METTL16 and inhibition of PD-1 synergistically suppressed in vivo growth of CRC cells.
    CONCLUSIONS: Our work identified the METTL16/PD-L1/PD-1 regulatory axis in CRC development and immune evasion, which represented a promising target for CRC treatment.
    Keywords:  METTL16; N6-methyladenosine; PD-L1; colorectal cancer; epigenetic regulation
  7. Nat Cell Biol. 2023 Aug 28.
      N6-methyladenosine (m6A) methylation can be deposited on chromatin-associated RNAs (caRNAs) by the RNA methyltransferase complex (MTC) to regulate chromatin state and transcription. However, the mechanism by which MTC is recruited to distinct genomic loci remains elusive. Here we identify RBFOX2, a well-studied RNA-binding protein, as a chromatin factor that preferentially recognizes m6A on caRNAs. RBFOX2 can recruit RBM15, an MTC component, to facilitate methylation of promoter-associated RNAs. RBM15 also physically interacts with YTHDC1 and recruits polycomb repressive complex 2 (PRC2) to the RBFOX2-bound loci for chromatin silencing and transcription suppression. Furthermore, we found that this RBFOX2/m6A/RBM15/YTHDC1/PRC2 axis plays a critical role in myeloid leukaemia. Downregulation of RBFOX2 notably inhibits survival/proliferation of acute myeloid leukaemia cells and promotes their myeloid differentiation. RBFOX2 is also required for self-renewal of leukaemia stem/initiation cells and acute myeloid leukaemia maintenance. Our study presents a pathway of m6A MTC recruitment and m6A deposition on caRNAs, resulting in locus-selective chromatin regulation, which has potential therapeutic implications in leukaemia.
  8. Heliyon. 2023 Aug;9(8): e18876
      As the most abundant internal mRNA modification, N6-methyladenosine (m6A) RNA methylation has been found to influence many biological events including bone mesenchymal stem cells (BMSCs) osteogenic differentiation. YTH N6-methyladenosine RNA binding protein C2 (YTHDC2) is an m6A reading protein with the ability to mediate the decay of combined methylated mRNA, however its role in BMSCs osteogenic differentiation remains unknown. In this study, we first found an increase of RUNX family transcription factor 2 (RUNX2) expression and a decrease of YTHDC2 expression during the process of BMSCs osteogenic differentiation. Furthermore, we transfected BMSCs with YTHDC2 interference fragment, resulting in an increased content of RUNX2 mRNA and protein inside BMSCs. Finally, through RNA Immunoprecipitation experiments, we confirmed that YTHDC2 protein can bind to RUNX2 mRNA and accelerate its decomposition. Moreover, the immunofluorescence staining also showed a negative correlation between YTHDC2 and RUNX2. In conclusion, during BMSCs osteogenic differentiation, YTHDC2 protein showed decreased expression, resulting in a higher level of RUNX2 (mRNA and protein) expression inside cells, indicating YTHDC2 as a promising molecular target for the regulation of BMSCs osteogenic differentiation.
    Keywords:  BMSCs; Osteogenic differentiation; RUNX2; YTHDC2; m6A
  9. Biol Direct. 2023 Sep 01. 18(1): 53
      BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common and challenging cancers in the world. N6-methyladenosine (m6A) modification and long non-coding RNAs (lncRNAs) play critical roles in the progression of HCC. However, there are few reports on genome-wide screening and functional annotations of m6A-methylated lncRNAs in HCC.METHODS: The expression levels of m6A methyltransferase METTL3 and the association with the prognosis in HCC were determined by RT-qPCR, public dataset platforms. Then, RNA-seq, Pearson correlation analysis, MeRIP-qPCR, RNA half-life assay, gene site-directed mutation, RIP assay and RT-qPCR analysis were employed to determine the downstream target of METTL3 in HCC. Subsequently, the expression levels and roles of lncRNA glucosylceramidase beta pseudogene 1 (GBAP1) in HCC were determined by Kaplan-meier curves, RT-qPCR, in vitro functional experiments and in vivo tumorigenesis and lung metastasis models. Then, the downstream target and pathway of GBAP1 were explored by GO biological process, KEGG pathway enrichment, luciferase reporter assay, RIP assay and rescue experiments and so on.
    RESULTS: METTL3 was upregulated in HCC and closely related to HCC prognosis. And METTL3 induced GBAP1 expression by acting as the m6A writer of GBAP1 and IGF2BP2 worked as its m6A reader. Clinically, GBAP1 expression was significantly associated with tumor size, venous infiltration, TNM stage and prognosis of HCC, Functionally, GBAP1 promoted HCC metastasis and growth both in vitro and in vivo. Furthermore, GBAP1 acted as the molecular sponge for miR-22-3p to increase the expression of bone morphogenetic protein receptor type 1A (BMPR1A), which then activated BMP/SMAD pathway in HCC cells.
    CONCLUSIONS: Our findings demonstrated that METTL3-induced GBAP1 promoted migration, invasion and proliferation of HCC cells via GBAP1/miR-22-3p/BMPR1A/SMAD axis. GBAP1 could be a potential prognosis indicator and therapeutic target for HCC.
    Keywords:  BMPR1A; GBAP1; Hepatocellular carcinoma; m6A; miR-22-3p
  10. Biomed Pharmacother. 2023 Aug 28. pii: S0753-3322(23)01196-4. [Epub ahead of print]166 115398
      Neuropathic pain (NP) is a common chronic pain condition resulted from lesions or diseases of somatosensory nervous system, but the pathogenesis remains unclear. A growing body of evidence supports the relationship between pathogenesis and N6-methyladenosine (m6A) modifications of RNA. However, studies on the role of m6A modifications in NP are still at an early stage. Elucidating different etiologies is important for understanding the specific pathogenesis of NP. This article provides a comprehensive review on the role of m6A methylation modifications including methyltransferases ("writers"), demethylases ("erasers"), and m6A binding proteins ("readers") in NP models. Further analysis of the pathogenic mechanism relationship between m6A and NP provided novel theoretical and practical significance for clinical treatment of NP.
    Keywords:  M6A methylation modification; Mechanism of action; Neuropathic pain; Rodent model; Targeted therapy
  11. STAR Protoc. 2023 Aug 25. pii: S2666-1667(23)00495-1. [Epub ahead of print]4(3): 102528
      Here, we present a detailed protocol to study the role of a human nuclear m6A RNA reader, YTHDC1, on chromatin-associated RNA targets. We describe steps for RNA extraction coupled to subnuclear fractionation to identify and study RNA-based regulations that take place in the chromatin-associated fraction. We then detail an RNA immunoprecipitation procedure adapted to identify chromatin-associated RNA targets. This protocol can be adapted to other human or mammalian chromatin-associated RNA binding proteins. For complete details on the use and execution of this protocol, please refer to Timcheva et al.1.
    Keywords:  Cell Biology; Cell separation/fractionation; Gene Expression; Molecular Biology; Protein expression and purification
  12. Sci Adv. 2023 Sep;9(35): eadg5234
      N6-methyladenosine (m6A) is the most abundant modification on messenger RNAs (mRNAs) and is catalyzed by methyltransferase-like protein 3 (Mettl3). To understand the role of m6A in a self-renewing somatic tissue, we deleted Mettl3 in epidermal progenitors in vivo. Mice lacking Mettl3 demonstrate marked features of dysfunctional development and self-renewal, including a loss of hair follicle morphogenesis and impaired cell adhesion and polarity associated with oral ulcerations. We show that Mettl3 promotes the m6A-mediated degradation of mRNAs encoding critical histone modifying enzymes. Depletion of Mettl3 results in the loss of m6A on these mRNAs and increases their expression and associated modifications, resulting in widespread gene expression abnormalities that mirror the gross phenotypic abnormalities. Collectively, these results have identified an additional layer of gene regulation within epithelial tissues, revealing an essential role for m6A in the regulation of chromatin modifiers, and underscoring a critical role for Mettl3-catalyzed m6A in proper epithelial development and self-renewal.
  13. Mol Cell. 2023 Aug 25. pii: S1097-2765(23)00649-4. [Epub ahead of print]
      N6-methyladenosine (m6A) RNA modification plays important roles in the governance of gene expression and is temporally regulated in different cell states. In contrast to global m6A profiling in bulk sequencing, single-cell technologies for analyzing m6A heterogeneity are not extensively established. Here, we developed single-nucleus m6A-CUT&Tag (sn-m6A-CT) for simultaneous profiling of m6A methylomes and transcriptomes within a single nucleus using mouse embryonic stem cells (mESCs). m6A-CT is capable of enriching m6A-marked RNA molecules in situ, without isolating RNAs from cells. We adapted m6A-CT to the droplet-based single-cell omics platform and demonstrated high-throughput performance in analyzing nuclei isolated from thousands of cells from various cell types. We show that sn-m6A-CT profiling is sufficient to determine cell identity and allows the generation of cell-type-specific m6A methylome landscapes from heterogeneous populations. These indicate that sn-m6A-CT provides additional dimensions to multimodal datasets and insights into epitranscriptomic landscape in defining cell fate identity and states.
    Keywords:  CUT&Tag; RNA modification; droplet-based; embryonic stem cell; epitranscriptomics; in situ; m(6)A; multimodal; single nucleus
  14. Theranostics. 2023 ;13(13): 4333-4355
      Rationale: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive solid tumor, with extremely low survival rates. Identifying key signaling pathways driving PDAC progression is crucial for the development of therapies to improve patient response rates. Kindlin-2, a multi-functional protein, is involved in numerous biological processes including cell proliferation, apoptosis and migration. However, little is known about the functions of Kindlin-2 in pancreatic cancer progression in vivo. Methods: In this study, we employ an in vivo PDAC mouse model to directly investigate the role of Kindlin-2 in PDAC progression. Then, we utilized RNA-sequencing, the molecular and cellular assays to determine the molecular mechanisms by which Kindlin-2 promotes PDAC progression. Results: We show that loss of Kindlin-2 markedly inhibits KrasG12D-driven pancreatic cancer progression in vivo as well as in vitro. Furthermore, we provide new mechanistic insight into how Kindlin-2 functions in this process, A fraction of Kindlin-2 was localized to the endoplasmic reticulum and associated with the RNA helicase DDX3X, a key regulator of mRNA translation. Loss of Kindlin-2 blocked DDX3X from binding to the 5'-untranslated region of c-Myc and inhibited DDX3X-mediated c-Myc translation, leading to reduced c-Myc-mediated glucose metabolism and tumor growth. Importantly, restoration of the expression of either the full-length Kindlin-2 or c-Myc, but not that of a DDX3X-binding-defective mutant of Kindlin-2, in Kindlin-2 deficient PDAC cells, reversed the inhibition of glycolysis and pancreatic cancer progression induced by the loss of Kindlin-2. Conclusion: Our studies reveal a novel Kindlin-2-DDX3X-c-Myc signaling axis in PDAC progression and suggest that inhibition of this signaling axis may provide a promising therapeutic approach to alleviate PDAC progression.
    Keywords:  DDX3X; Kindlin-2; Pancreatic ductal adenocarcinoma; c-Myc; translation
  15. Aging Cell. 2023 Aug 28. e13972
      N6 -methyladenosine (m6 A) is a dynamic and reversible RNA modification that has emerged as a crucial player in the life cycle of RNA, thus playing a pivotal role in various biological processes. In recent years, the potential involvement of RNA m6 A modification in aging and age-related diseases has gained increasing attention, making it a promising target for understanding the molecular mechanisms underlying aging and developing new therapeutic strategies. This Perspective article will summarize the current advances in aging-related m6 A regulation, highlighting the most significant findings and their implications for our understanding of cellular senescence and aging, and the potential for targeting RNA m6 A regulation as a therapeutic strategy. We will also discuss the limitations and challenges in this field and provide insights into future research directions. By providing a comprehensive overview of the current state of the field, this Perspective article aims to facilitate further advances in our understanding of the molecular mechanisms underlying aging and to identify new therapeutic targets for aging-related diseases.
    Keywords:  RNA methylation; aging; disease; m6A; senescence
  16. J Clin Invest. 2023 Aug 29. pii: e170173. [Epub ahead of print]
      PCIF1 can mediate the methylation of N6,2'-O-dimethyladenosine (m6Am) in mRNA. Yet, the detailed interplay between PCIF1 and the potential cofactors and its pathological significance remains elusive. Here, we demonstrated that PCIF1-mediated cap mRNA m6Am modification promoted head and neck squamous cell carcinoma (HNSCC) progression both in vitro and in vivo. CTBP2 was identified as a cofactor of PCIF1 to catalyze m6Am deposition on mRNA. CLIP-seq data demonstrated CTBP2 bound to similar mRNAs as PCIF1. We then utilized m6Am-seq method to profile mRNA m6Am site at single-base resolution and found mRNA of TET2, a well-known tumor suppressor, was a major target substrate of PCIF1-CTBP2 complex. Mechanistically, knockout of CTBP2 reduced PCIF1 occupancy on TET2 mRNA and PCIF1-CTBP2 complex negatively regulated the translation of TET2 mRNA. Collectively, our study demonstrated the oncogenic function of the epitranscriptome regulator PCIF1-CTBP2 complex, highlighting the importance of the m6Am modification in tumor progression.
    Keywords:  Cell Biology; Epigenetics; Head and neck cancer; Oncology
  17. Mol Carcinog. 2023 Aug 29.
      Abnormal RNA N7-methylguanosine (m7G) modification is known to contribute to effects on tumor occurrence and development. Nevertheless, the mechanisms of its function in immunoregulation, tumor microenvironment (TME) modulation, and tumor promotion remain largely unknown. A series of computer-aided bioinformatic analyses were conducted based on transcriptomic, single-cell sequence, and spatial transcriptomic data to determine the m7G modification patterns in head and neck squamous cell carcinoma (HNSCC). Consensus clustering approach was employed according to the expressions of 33 m7G regulators. ESTIMATE, CIBERSORT, and single sample gene set enrichment analysis algorithms were adopted to investigate the immune cell infiltration features. A prognostic model named m7Gscore was established. Seurat, SingleR, and Monocle2 were used to analyze the single-cell sequence profiling. STUtility was used to integrate multiple spatial transcriptomic datasets. Quantitative reverse transcription polymerase chain reaction, transwell, and wound-healing assay were performed to verify the oncogenes. Here, three different m7G modification patterns were highlighted in HNSCC patients, which were also related to various clinical manifestations and three representative immunophenotypes: immune-excluded, immune-desert, and inflamed, separately. Patients with lower m7Gscore were highlighted by higher immune cell infiltrations, better overall survival rates, lesser tumor mutation burden (TMB), lower sensitivities to target inhibitors therapies, and better immunotherapeutic response. Moreover, DCPS, EIF4E, EIF4E2, LSM1, NCBP2, NUDT1, and NUDT5 were identified to play critical roles in T-cell differentiation. Knockdown of LSM1/NUDT5 could restrain the malignancy of HNSCC cells. Collectively, quantitative assessment of m7G modification patterns in individual HNSCC patients could contribute to identifying more efficient immunotherapeutic approaches and improve the clinical outcome of HNSCC.
    Keywords:  HNSCC; LSM1; NUDT5; immunotherapy; m7G modification; prognosis
  18. Cell Death Differ. 2023 Aug 30.
      The treatment options for advanced papillary thyroid cancer (PTC) and anaplastic thyroid cancer (ATC) refractory to standard therapies are limited. Although anti-PD-1 therapy has a manageable safety profile and has been effective in a small percentage of patients with advanced PTC and refractory ATC, the majority of the patients either do not respond or develop resistance to anti-PD-1 therapy. N6-methyladenosine (m6A) modification is a critical determinant of the complexity of the tumor microenvironment (TME). However, it is unclear whether and how m6A modification in tumor cells shapes the immune landscape of PTC and ATC. In this study, we performed bulk and single cell RNA sequencing analysis of PTC and ATC tissues, and found that low METTL3 expression not only correlated to poor response to immune checkpoint blockade (ICB) but was also associated with increased TNF family-related ligand-receptor interactions in the immunosuppressive Tregs and exhausted T cells. Furthermore, overexpression of METTL3 in PTC and ATC cells enhanced the efficacy of anti-PD-1 therapy in a peripheral blood mononuclear cell humanized NCG (huPBMC-NCG) mouse model. Mechanistically, M2 macrophage-derived extracellular vesicles (M2 EVs) inhibited METTL3 expression in PTC and ATC cells via miR-21-5p. Downregulation of METTL3 promoted demethylation of CD70 mRNA, which prevented YTHDF2-mediated degradation of the transcripts. The stabilization of CD70 mRNA, and the subsequent upregulation in CD70 protein levels increased the abundance of the immunosuppressive Tregs and terminally exhausted T cells, thereby inducing resistance to anti-PD-1 therapy. Furthermore, blocking CD70 using cusatuzumab, a high-affinity monoclonal antibody, reversed the anti-PD-1 therapy resistance induced by M2 EVs in vivo. Finally, we demonstrated that METTL3 expression negatively correlated with CD70 expression and M2 macrophages and Tregs infiltration in PTC and ATC tissues. Our findings provide new insights into developing novel therapies for advanced PTC and ATC.
  19. Exp Hematol Oncol. 2023 Aug 29. 12(1): 75
      BACKGROUND: The mechanisms underlying the occurrence and development of esophageal squamous cell carcinoma (ESCC) remains to be elucidated. The present study aims to investigate the roles and implications of IGF2BP1 overexpression in ESCC.METHODS: IGF2BP1 protein expression in ESCC samples was assessed by immunohistochemistry (IHC), and the mRNA abundance of IGF2BP1 and INHBA was analyzed with TCGA datasets and by RNA in situ hybridization (RISH). The methylation level of the IGF2BP1 promoter region was detected by methylation-specific PCR (MSP-PCR). Cell viability, migration, invasion and in vivo metastasis assays were performed to explore the roles of IGF2BP1 overexpression in ESCC. RNA immunoprecipitation sequencing (RIP-seq) and mass spectrometry were applied to identify the target RNAs and interacting proteins of IGF2BP1, respectively. RIP-PCR, RNA pulldown, immunofluorescence (IF), gene-specific m6A PCR and RNA stability assays were used to uncover the molecular mechanisms underlying the malignant phenotypes of ESCC cells caused by IGF2BP1 dysregulation. BTYNB, a small molecular inhibitor of IGF2BP1, was evaluated for its inhibitory effect on the malignant phenotypes of ESCC cells.
    RESULTS: IGF2BP1 overexpression was detected in ESCC tissues and associated with the depth of tumor invasion. In addition, IGF2BP1 mRNA expression in ESCC cells was negatively correlated with the level of its promoter methylation. Knockdown of IGF2BP1 inhibited ESCC cell invasion and migration as well as tumor metastasis. Mechanistically, we observed that IGF2BP1 bound and stabilized INHBA mRNA and then resulted in higher protein expression of INHBA, leading to the activation of Smad2/3 signaling, thus promoting malignant phenotypes. The mRNA level of INHBA was upregulated in ESCC tissues as well. Furthermore, IGF2BP1 interacted with G3BP stress granule assembly factor 1 (G3BP1). Knockdown of G3BP1 also down-regulated the INHBA-Smad2/3 signaling. BTYNB abolished this activated signaling and significantly attenuated the malignant phenotypes of ESCC cells.
    CONCLUSIONS: Elevated expression of IGF2BP1 is a frequent event in ESCC tissues and might be a candidate biomarker for the disease. IGF2BP1 overexpression promotes the invasion and migration of ESCC cells by activating the INHBA-Smad2/3 pathway, providing a potential therapeutic target for ESCC patients with high expression of IGF2BP1.
    Keywords:  Esophageal squamous cell carcinoma; IGF2BP1; INHBA; Invasion; Migration; RNA binding protein
  20. Genes Environ. 2023 Sep 01. 45(1): 23
      BACKGROUND: Evidence showed that N6-methyladenosine (m6A) is strongly associated with male germline development. However, the role of m6A methylation on circRNAs in amphibians remains unknown. In this study, we conducted m6A sequencing analysis to explore the m6A transcriptome-wide profile of circRNAs in testis tissues of Xenopus laevis (X. laevis) with and without treatment with 100 µg/L atrazine (AZ).RESULTS: The analysis showed that m6A modification of circRNAs enriched in sense overlapping in testes of X. laevis. We identified the differential m6A modification sites within circRNAs in testes of AZ-exposed X. laevis and compared that with animals from control group. The results showed that a total of 1507 methylated m6A sites was induced by AZ (760 up-methylated and 747 down-methylated). The cross-analysis exhibited a negative correlation of differentially methylated m6A peaks and circRNAs expression level. The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated that 20 key pathways may be involved in the mechanism of testis damage of AZ-exposed X. laevis.
    CONCLUSIONS: These findings indicated that differentially m6A-methylated circRNAs may play important roles in abnormal testis development of AZ-exposed X. laevis. This study is the first report about a map of m6A modification of circRNAs in male X. laevis and provides a basis for further studying on the function and mechanism of m6A methylation of circRNAs in the testis development of amphibian.
    Keywords:  Amphibious; Atrazine; CircRNA; M6A; RNA methylation
  21. Front Immunol. 2023 ;14 1152806
      Rationale: RNA modifications, containing m6A, m1A, alternative polyadenylation and adenosine-to-inosine RNA editing, involve in critical cancerous immunity and cancerous processes. However, the functional roles of RNA modification writers in bladder cancer (BLCA) are largely unknown.Methods: In this study, unsupervised clustering was used to identify novel RNA modification writers -mediated molecular subtypes in BLCA. A corresponding quantitative indicator called WriterScore was developed using univariate Cox and Least absolute shrinkage and selection operator (LASSO) analysis. Then, we systematically analyzed the correlation between RNA modification writer-related clusters (WriterScore) and immunological characteristics, classical molecular subtypes, clinicopathologic features and treatment options in BLCA. Finally, we validated the WriterScore in multiple other external BLCA datasets, clinical sample dataset in Shengjing Hospital and pancancer.
    Results: Two RNA modification writer-related clusters and three DEGclusters were obtained. These RNA modification writer-related clusters (WriterScore) were strongly associated with immunological characteristics, classical molecular subtypes, clinicopathologic features of BLCA. Moreover, WriterScore can properly predict the clinical outcomes and immunotherapy of BLCA patients.
    Conclusion: Our study systematically investigated the role of RNA modification writers and developed a significant WriterScore to guide several treatment options in BLCA, which might bring some potential benefits for BLCA patients.
    Keywords:  RNA modification writers; bladder cancer; immunotherapy benefits; tumor microenvironment; unsupervised clustering
  22. Curr Top Med Chem. 2023 Aug 25.
      Cardiac fibrosis is known as the expansion of the cardiac interstitium through excessive deposition of extracellular matrix proteins; this process is performed by a multifunctional cell known as the cardiac fibroblast. After the myocardial injury, these cells are activated as a repair program, increase, and switch to a contractile phenotype, which is evidenced by an increase in alpha-smooth muscle actin. Likewise, there is an increase in type I and III collagen, which are considered profibrotic biomarkers. It is believed that one of the proteins involved in cardiac remodeling is METTL3, which is the enzyme responsible for N6-methyladenosine (m6A) methylation, the most common and abundant epigenetic modification of eukaryotic mRNA. This review focuses on recent studies in which the possible role of METTL3 in the progression of fibrosis has been demonstrated, mainly in cardiac fibrogenesis.
    Keywords:  METTL3; cardiac fibrosis.; cardiac myofibroblasts; cardiac remodeling
  23. Clin Transl Med. 2023 Sep;13(9): e1361
      BACKGROUND: Super enhancers (SE) play pivotal roles in cell identity and diseases occur including tumorigenesis. The depletion of SE-associated lncRNA transcripts, also known as super-lncRNA, causes the activity of SE to be dysregulated.METHODS: We screened and identified an elevated metastasis-associated SE-lncRNA SUCLG2-AS1 in nasopharyngeal carcinoma (NPC) using RNA-sequencing, real-time quantitative polymerase chain reaction (RT-qPCR) and bioinformatics. Western blotting, RT-qPCR, methylated RNA immunoprecipitation (MeRIP), RNA immunoprecipitation, chromatin immunoprecipitation, RNA pull-down and 3C (chromosome conformation capture assays) were used for mechanistic studies.
    RESULTS: SUCLG2-AS1 was correlated with a poor prognosis. SUCLG2-AS1 promotes NPC cell invasion and metastasis while repressing apoptosis and radiosensitivity in vitro and in vivo. Mechanistically, high SUCLG2-AS1 expression occurred in an m6A-dependent manner. SUCLG2-AS1 was found to be located in the SE region of SOX2, and it regulated the expression of SOX2 via long-range chromatin loop formation, which via mediating CTCF (transcription factor) occupied the SE and promoter region of SOX2, thus regulating the metastasis and radiosensitivity of NPC.
    CONCLUSIONS: Taken together, our data suggest that SUCLG2-AS1 may serve as a novel intervention target for the clinical treatment of NPC.
    Keywords:  SOX2; SUCLG2-AS1; nasopharyngeal carcinoma; radiosensitivity; super-enhancer
  24. Cancer Biol Ther. 2023 Dec 31. 24(1): 2249174
      Infection with high-risk human papillomavirus (HPV), for example, with types 16 and 18, is closely associated with cervical cancer development, which continues to threaten women's health globally. Although HPV oncogenes have been recognized as the main cause of transformation of normal cervical epithelial cells, non-coding RNA could also be involved in the initiation and promotion of cervical cancer development. Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), a well-documented long non-coding RNA (lncRNA), has been previously reported to exert roles in HPV-positive cervical cancer; however, the detailed underlying mechanism has yet to be investigated. In the present study, high expression levels of MALAT1 in HPV-Positive Cervical Cancer cells were confirmed, and silencing MALAT1 resulted in decreased rates of cell proliferation, migration, and invasion, both in vitro and in a zebrafish xenograft tumor model. Moreover, the results obtained showed that silencing MALAT1 led to down-regulation of the N6-methyladenosine (m6A) demethylase ALKBH5 via regulating miR-141-3p expression, which caused a decrease in the expression levels of matrix metalloproteinase 2 (MMP2) and MMP9 expression, thereby suppressing cell migration and invasion. Taken together, the results obtained have suggested that the MALAT-ALKBH5 signaling axis may be activated in HPV-positive cervical cancer cells, which could contribute to cell proliferation and metastasis through the regulation of key genes, such as MMP2 or MMP9. The findings of the present study should both help to improve our understanding of the underlying tumorigenic mechanisms of HPV-positive cervical cancer and be of further use in the development of potential therapeutic drugs.
    Keywords:  ALKBH5; HPV-positive cervical cancer; MALAT1; invasion; migration; proliferation
  25. J Cancer Res Clin Oncol. 2023 Aug 30.
      Hepatocellular carcinoma (HCC), featured with high prevalence and poor prognosis, is the major cause of cancer-related deaths worldwide. As a subgroup of liver cancer cells capable of differentiation, tumorigenesis and self-renewal, liver cancer stem cells (LCSCs) serve as one of the reasons leading to HCC progression and therapeutic resistance. Therefore, in-depth exploration of novel molecular biomarkers related to LSCSs is of great necessity. In our study, we found that human AlkB homolog H5 (ALKBH5) expression was enriched in LCSCs, which could foster proliferation, invasion and migration of the HCC cells. Mechanically, ALKBH5 positively mediated the expression of SOX4 via demethylation, and SOX4 promoted SHH expression at the transcriptional level to activate sonic hedgehog (SHH) signaling pathway. Furthermore, exosomes derived from CD133+ HCC cells could transmit ALKBH5 into THP-1 cells, which might be associated with M2 polarization of macrophages. In summary, the ALKBH5/SOX4 axis plays a significant role in exacerbating LCSC properties via activating SHH signaling pathway, and ALKBH5 could be a critical effector related to macrophage M2 polarization. These findings might provide a promising new biomarker for HCC diagnosis and treatment.
    Keywords:  ALKBH5; Exosome; Liver cancer stem cell; Macrophage polarization; N6-methyladenosine modification; SHH signaling pathway
  26. Front Immunol. 2023 ;14 1225023
      Background: Both lactylation and m6A modification have important implications for the development of clear cell renal cell carcinoma (ccRCC), and we aimed to use crosstalk genes of both to reveal the prognostic and immunological features of ccRCC.Methods: Our first step was to look for lactylation-related genes that differed between normal and tumor tissues, and then by correlation analysis, we found the genes associated with M6A. Following that, ccRCC subtypes will be identified and risk models will be constructed to compare the prognosis and tumor microenvironment among different subgroups. A nomogram was constructed to predict the prognosis of ccRCC, and in vitro, experiments were conducted to validate the expression and function of key genes.
    Results: We screened 100 crosstalk genes and identified 2 ccRCC subtypes. A total of 11 prognostic genes were screened for building a risk model. we observed higher immune scores, elevated tumor mutational burden, and microsatellite instability scores in the high-risk group. Therefore, individuals classified as high-risk would derive greater benefits from immunotherapy. The nomogram's ability to predict overall survival with a 1-year AUC of 0.863 demonstrates its significant practical utility. In addition, HIBCH was identified as a potential therapeutic target and its expression and function were verified by in vitro experiments.
    Conclusion: In addition to developing a precise prognostic nomogram for patients with ccRCC, our study also discovered the potential of HIBCH as a biomarker for the disease.
    Keywords:  M6A; clear cell renal cell carcinoma; immunotherapy; lactylation; tumor microenvironment
  27. J Biol Chem. 2023 Aug 24. pii: S0021-9258(23)02223-8. [Epub ahead of print] 105195
      The regulation of translation provides a rapid and direct mechanism to modulate the cellular proteome. In eukaryotes, an established model for the recruitment of ribosomes to mRNA depends upon a set of conserved translation initiation factors. Nevertheless, how cells orchestrate and define the selection of individual mRNAs for translation, as opposed to other potential cytosolic fates, is poorly understood. We have previously found significant variation in the interaction between individual mRNAs and an array of translation initiation factors. Indeed, mRNAs can be separated into different classes based upon these interactions to provide a framework for understanding different modes of translation initiation. Here, we extend this approach to include new mRNA interaction profiles for additional proteins involved in shaping the cytoplasmic fate of mRNAs. This work defines a set of seven mRNA clusters, based on their interaction profiles with 12 factors involved in translation and/or RNA binding. The mRNA clusters share both physical and functional characteristics to provide a rationale for the interaction profiles. Moreover, a comparison with mRNA interaction profiles from a host of RNA binding proteins (RBPs) suggests that there are defined patterns in the interactions of functionally related mRNAs. Therefore, this work defines global cytoplasmic mRNA binding modules that likely coordinate the synthesis of functionally related proteins.
    Keywords:  RIP-sequencing (RIP-seq); RNA binding proteins; mRNA; mRNA decay; translation initiation
  28. J Exp Clin Cancer Res. 2023 Sep 01. 42(1): 222
      BACKGROUND: FAT4 (FAT Atypical Cadherin 4) is a member of the cadherin-associated protein family, which has been shown to function as a tumor suppressor by inhibiting proliferation and metastasis. The Wnt/β-catenin pathway activation is highly associated with PD-L1-associated tumor immune escape. Here, we report the mechanism by which FAT4 overexpression regulates anti-tumor immunity in cervical cancer by inhibiting PD-L1 N-glycosylation and cell membrane localization in a β-catenin-dependent manner.METHODS: FAT4 expression was first detected in cervical cancer tissues and cell lines. Cell proliferation, clone formation, and immunofluorescence were used to determine the tumor suppressive impact of FAT4 overexpression in vitro, and the findings were confirmed in immunodeficient and immunocomplete mice xenografts. Through functional and mechanistic experiments in vivo and in vitro, we investigated how FAT4 overexpression affects the antitumor immunity via the β-catenin/STT3/PD-L1 axis.
    RESULTS: FAT4 is downregulated in cervical cancer tissues and cell lines. We determined that FAT4 binds to β-catenin and antagonizes its nuclear localization, promotes phosphorylation and degradation of β-catenin by the degradation complexes (AXIN1, APC, GSK3β, CK1). FAT4 overexpression decreases programmed death-ligand 1 (PD-L1) mRNA expression at the transcriptional level, and causes aberrant glycosylation of PD-L1 via STT3A at the post-translational modifications (PTMs) level, leading to its endoplasmic reticulum (ER) accumulation and polyubiquitination-dependent degradation. We found that FAT4 overexpression promotes aberrant PD-L1 glycosylation and degradation in a β-catenin-dependent manner, thereby increasing cytotoxic T lymphocyte (CTL) activity in immunoreactive mouse models.
    CONCLUSIONS: These findings address the basis of Wnt/β-catenin pathway activation in cervical cancer and provide combination immunotherapy options for targeting the FAT4/β-catenin/STT3/PD-L1 axis. Schematic cartoons showing the antitumor immunity mechanism of FAT4. (left) when Wnts bind to their receptors, which are made up of Frizzled proteins and LRP5/6, the cytoplasmic protein DVL is activated, inducing the aggregation of degradation complexes (AXIN, GSK3β, CK1, APC) to the receptor. Subsequently, stable β-catenin translocates into the nucleus and binds to TCF/LEF and TCF7L2 transcription factors, leading to target genes transcription. The catalytically active subunit of oligosaccharyltransferase, STT3A, enhances PD-L1 glycosylation, and N-glycosylated PD-L1 translocates to the cell membrane via the ER-to-Golgi pathway, resulting in immune evasion. (Right) FAT4 exerts antitumor immunity mainly through following mechanisms: (i) FAT4 binds to β-catenin and antagonizes its nuclear localization, promotes phosphorylation and degradation of β-catenin by the degradation complexes (AXIN1, APC, GSK3β, CK1); (ii) FAT4 inhibits PD-L1 and STT3A transcription in a β-catenin-dependent manner and induces aberrant PD-L1 glycosylation and ubiquitination-dependent degradation; (iii) Promotes activation of cytotoxic T lymphocytes (CTL) and infiltration into the tumor microenvironment.
    Keywords:  CTL; Cervical cancer; FAT4; PD-L1; Wnt/β-catenin pathway
  29. Biochim Biophys Acta Mol Basis Dis. 2023 Aug 26. pii: S0925-4439(23)00223-5. [Epub ahead of print] 166857
      Alzheimer's Disease (AD) is a neurodegenerative disorder characterized by accumulation of β-amyloid aggregates and loss of proteostasis. Transfer RNA (tRNA) modifications play a crucial role in maintaining proteostasis, but their impact in AD remains unclear. Here, we report that expression of the tRNA modifying enzyme ELP3 is reduced in the brain of AD patients and amyloid mouse models and negatively correlates with amyloid plaque mean density. We further show that SH-SY5Y neuronal cells carrying the amyloidogenic Swedish familial AD mutation (SH-SWE) display reduced ELP3 levels, tRNA hypomodifications and proteostasis impairments when compared to cells not carrying the mutation (SH-WT). Additionally, exposing SH-WT cells to the secretome of SH-SWE cells led to reduced ELP3 expression, wobble uridine tRNA hypomodification, and increased protein aggregation. Importantly, correcting tRNA deficits due to ELP3 reduction reverted proteostasis impairments. These findings suggest that amyloid pathology dysregulates proteostasis by reducing ELP3 expression and tRNA modification levels, and that targeting tRNA modifications may be a potential therapeutic avenue to restore neuronal proteostasis in AD and preserve neuronal function.
    Keywords:  Alzheimer's disease; Elongator complex subunit 3 (ELP3); Proteostasis; Translation; tRNA modifications
  30. Cell Death Discov. 2023 Aug 29. 9(1): 324
      Cutaneous wound healing, an integral part for protection of skin barrier, is a complex biological process and intimately associated with keratinocyte migration. However, mechanisms regulating keratinocyte migration in the process of cutaneous wound repair remain largely unknown. Here, we found that N-acetyltransferase 10 (NAT10) is essential for cutaneous wound repair in an in vivo skin wound healing model-a significant delay of wound repair in Nat10 haploinsufficient mice and a remarkable inhibition of keratinocyte migration by NAT10 knockdown in an in vitro keratinocyte migration model. We further demonstrate that loss of NAT10 expression attenuates the wound-induced IL-6/IL-8 expression through inhibiting NF-κB/p65 activity in keratinocytes. By deeply digging, silencing NAT10 compromises the level of nuclear p65 by facilitating its poly-ubiquitination, thus accelerates its degradation in the nucleus. Notably, we detected a strong positive correlation between the expression of NAT10 and relevant NF-kB/p65-IL6 signaling activity in mouse wound skin tissues. Overall, our study reveals an important role of NAT10 on cutaneous wound repair by potentiating NF-κB/p65-IL-6/8-STAT3 signaling. Targeting NAT10 might be a potential strategy for the treatment of skin wound dysfunctions and related diseases.
  31. Anticancer Res. 2023 Sep;43(9): 3961-3968
      BACKGROUND/AIM: Forkhead box M1 (FOXM1) is a transcription factor closely associated with various human malignancies and is considered an attractive target for cancer therapy. Mesothelioma is a malignancy primarily due to asbestos exposure and certain genetic factors, requiring a better understanding of tumorigenesis for improved treatment. Asbestos-exposed human mesothelial cells have been reported to up-regulate FOXM1 expression in a dose-dependent manner.MATERIALS AND METHODS: FOXM1 expression was evaluated in mesothelioma tissues and cell lines. FOXM1 small interfering RNA was transfected into mesothelioma cell lines to analyze its biological functions and regulatory mechanisms.
    RESULTS: FOXM1 was over-expressed in mesothelioma tissues and cell lines. Knock-down of FOXM1 in mesothelioma cell lines inhibited cell proliferation, migration, and invasion. These results suggest that up-regulation of FOXM1 expression promotes mesothelioma tumorigenesis and progression. We previously reported that insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3) promotes the proliferation, migration, and invasion of mesothelioma cell lines. In this study, IGF2BP3 knock-down suppressed FOXM1 expression in mesothelioma cell lines. Our results suggest that IGF2BP3, an upstream regulator, contributes to increased FOXM1 expression. Furthermore, IGF2BP3 and FOXM1 knock-down suppressed SMAD signaling by inhibiting SMAD2/3 phosphorylation in mesothelioma cell lines.
    CONCLUSION: IGF2BP3/FOXM1 promotes mesothelioma cell migration and invasion via SMAD signaling, highlighting IGF2BP3/FOXM1 as a potential target for mesothelioma treatment.
    Keywords:  FOXM1; IGF2BP3; Mesothelioma; SMAD; invasion; migration