bims-rimeca Biomed News
on RNA methylation in cancer
Issue of 2022–05–08
28 papers selected by
Sk Ramiz Islam, Saha Institute of Nuclear Physics



  1. Exp Cell Res. 2022 Apr 27. pii: S0014-4827(22)00169-0. [Epub ahead of print] 113176
      The N6-methyladenosine (m6A) is involved in the regulation of cell proliferation and metastasis formation in multiple cancers. However, the biological significance of RNA m6A reader IGF2BP1 and the modification of IGF2BP1 itself have not been fully investigated. Here, we analyzed the functions and mechanism of IGF2BP1 in gastric cancer (GC). Results showed that IGF2BP1 upregulated in GC tissue and acted as a predictor of poor prognosis for GC patients. Functionally, IGF2BP1 promoted the migration and aerobic glycolysis of GC cells in vitro. Moreover, IGF2BP1 knockdown repressed the tumor growth in vivo. We also demonstrated that IGF2BP1 directly interacted with c-MYC mRNA via m6A-dependent manner to by stabilize its stability. Overall, these findings demonstrated that m6A reader IGF2BP1 facilitated the carcinogenic of GC in m6A/c-Myc-dependent manner, which might provide critical therapeutic strategy for GC.
    Keywords:  Aerobic glycolysis; Gastric cancer; IGF2BP1; N(6)-methyladenosine; c-Myc
    DOI:  https://doi.org/10.1016/j.yexcr.2022.113176
  2. Cancer Res. 2022 May 03. 82(9): 1789-1802
      The RNA N6-methyladenosine (m6A) writer methyltransferase-like 3 (METTL3) is upregulated in many types of cancer and promotes cancer progression by increasing expression of several oncogenes. Therefore, a better understanding of the mechanisms regulating METTL3 expression and the key targets of METTL3 in cancer cells could provide new therapeutic targets. In this study, we found that activated JNK signaling is associated with increased METTL3 expression in bladder cancer. Knockdown of JNK1 or administration of a JNK inhibitor impaired the binding of c-Jun with the METTL3 promoter, thereby decreasing the expression of METTL3 and global RNA m6A levels. Moreover, RNA m6A sequencing indicated enrichment of m6A in the 3'-UTR of immune checkpoint PD-L1 mRNA, which could be recognized by the m6A reader IGF2BP1 to mediate RNA stability and expression levels of PD-L1. Inhibition of JNK signaling suppressed m6A abundance in PD-L1 mRNA, leading to decreased PD-L1 expression. Functionally, METTL3 was essential for bladder cancer cells to resist the cytotoxicity of CD8+ T cells by regulating PD-L1 expression. Additionally, JNK signaling contributed to tumor immune escape in a METTL3-dependent manner both in vitro and in vivo. These data reveal the JNK/METTL3 axis as a mechanism of aberrant m6A modification and immune regulation in bladder cancer.
    SIGNIFICANCE: The identification of a novel m6A-dependent mechanism underlying immune system evasion by bladder cancer cells reveals JNK signaling as a potential target for bladder cancer immunotherapy.
    DOI:  https://doi.org/10.1158/0008-5472.CAN-21-1323
  3. EBioMedicine. 2022 Apr 28. pii: S2352-3964(22)00203-1. [Epub ahead of print]80 104019
       BACKGROUND: N6-methyladenosine (m6A) is the most common and abundant mRNA modification and it plays crucial roles in many biological processes. However, as a key RNA demethylase, alkylation repair homolog protein 5 (ALKBH5) has not been well studied in human osteosarcoma. The present study sought to explore ALKBH5-mediated m6A modification and the underlying mechanisms in human osteosarcoma.
    METHODS: The expression of ALKBH5 and its correlation with clinicopathological features were examined by bioinformatics analysis and tissue microarrays. Cellular proliferation was detected by CCK8 assays. Cell cycle and apoptosis were analyzed by TUNEL and Flow cytometry assay. Finally, investigation of the regulatory mechanism of ALKBH5 in human osteosarcoma was performed by MeRIP assay, RNA-sequencing, dual luciferase reporter assay, RNA pull-down and RNA stability assay. Tumor xenograft models were established for in vivo experiments.
    FINDINGS: Our data showed that low expression of ALKBH5 was associated with worse overall survival for osteosarcoma patients. Reducing m6A mRNA levels in human osteosarcoma cells through ALKBH5 up-regulation lead to cell proliferation inhibition, cell apoptosis and cycle arrest. We identified SOCS3, a negative regulator of STAT3, as a downstream target of ALKBH5-mediated m6A modification. And the m6A modified SOCS3 mRNA was recognized by YTHDF2, which promotes the decay of SOCS3. Mechanistically, our data revealed that ALKBH5 inactivated STAT3 pathway by increasing SOCS3 expression via an m6A-YTHDF2-dependent manner.
    INTERPRETATION: M6A methylation is rising as a pathway affecting tumorigenicity and tumor progression. Our findings illuminate the clinical significance of ALKBH5-mediated m6A modification in human osteosarcoma and the regulatory mechanisms underlying tumor proliferation and growth, suggesting that ALKBH5 is a potential biomarker for treatment in human osteosarcoma.
    FUNDING: This work was supported by and Science and Technology foundation of Hubei, China (Grant No.2017CFB762); the Tongji hospital foundation (Grant No.2201103013); and the National Natural Science Foudation of China (No.82002849).
    Keywords:  ALKBH5; Osteosarcoma; SOCS3; STAT3; YTHDF2; m6A
    DOI:  https://doi.org/10.1016/j.ebiom.2022.104019
  4. Cytokine Growth Factor Rev. 2022 Apr 22. pii: S1359-6101(22)00026-0. [Epub ahead of print]
      RNA N6-methyladenosine (m6A) modification is abundant in eukaryotes, bacteria and archaea. It is an RNA modification mainly existing in messenger RNA (mRNAs) and has a significant effect on the metabolism and function of mRNAs. m6A modification is controlled by three types of proteins, namely methyltransferase as the "writers", demethylase as the "erasers", and specific m6A recognized protein (YTHDF1-3) as the "readers". Recent studies have shown that m6A modification plays an important role in cancer, viral infection and autoimmune diseases. In this review, we will elaborate on the m6A modifications in the homeostasis and differentiation of T cells. Then we will further summarize the effects of m6A modification on the T cell responses and T cell-mediated autoimmune diseases. This will advance T cell epigenetics research and provide potential biomarkers and therapeutic targets for autoimmune diseases.
    Keywords:  Autoimmunity; Differentiation; T cell responses; m(6)A machinery
    DOI:  https://doi.org/10.1016/j.cytogfr.2022.04.004
  5. Bioengineered. 2022 May;13(5): 11541-11550
      N6-methyladenosine (m6A) modification acts as the most prevalent internal modification in eukaryotic mRNA. Emerging evidence shows the critical biological roles of m6A key enzymes in human cancers. However, the roles of m6A binding protein IGF2BP2 in gastric cancer (GC) progression are still unclear. In this study, we confirmed that IGF2BP2 was highly expressed in GC cell lines and tumor tissues. Knocking down of IGF2BP2 suppressed cell proliferation and migration, and repressed xenograft tumor growth in vivo, while IGF2BP2 overexpression promoted the proliferation and migration. Mechanistically, we identified that IGF2BP2 regulated GC the proliferation/migration through recognizing the m6A modification sites of SIRT1 mRNA. In general, our findings demonstrated a novel regulatory mechanism that IGF2BP2/SIRT1 axis modulated GC progression in an m6A-dependent manner, suggesting that m6A may be a therapeutic target for GC.
    Keywords:  Gastric cancer; IGF2BP2; N6-methyladenosine; SIRT1
    DOI:  https://doi.org/10.1080/21655979.2022.2068920
  6. Elife. 2022 May 03. pii: e75231. [Epub ahead of print]11
      METTL3 and N6-methyladenosine (m6A) are involved in many types of biological and pathological processes, including DNA repair. However, the function and mechanism of METTL3 in DNA repair and chemotherapeutic response remain largely unknown. In present study, we identified that METTL3 participates in the regulation of homologous recombination repair (HR), which further influences chemotherapeutic response in both MCF-7 and MDA-MB-231 breast cancer (BC) cells. Knockdown of METTL3 sensitized these BC cells to Adriamycin (ADR; also named as doxorubicin) treatment and increased accumulation of DNA damage. Mechanically, we demonstrated that inhibition of METTL3 impaired HR efficiency and increased ADR-induced DNA damage by regulating m6A modification of EGF/RAD51 axis. METTL3 promoted EGF expression through m6A modification, which further upregulated RAD51 expression, resulting in enhanced HR activity. We further demonstrated that the m6A 'reader', YTHDC1, bound to the m6A modified EGF transcript and promoted EGF synthesis, which enhanced HR and cell survival during ADR treatment in breast cancer cells. Our findings reveal a pivotal mechanism of METTL3-mediated HR and chemotherapeutic drug response, which may contribute to cancer therapy.
    Keywords:  biochemistry; cell biology; chemical biology; mouse
    DOI:  https://doi.org/10.7554/eLife.75231
  7. Apoptosis. 2022 May 03.
      Hepatocellular carcinoma (HCC) is insidious and prone to metastasis and recurrence. Currently, no effective treatment is available for HCC. Furthermore, HCC does not respond to various radio- and chemotherapies, and the molecular mechanism of treatment resistance is unclear. Here, we found that p53 n6-methyladenosine (m6A) played a decisive role in regulating HCC sensitivity to chemotherapy via the p53 activator RG7112 and the vascular endothelial growth factor receptor inhibitor apatinib. Our results reveal that p53 activation plays a crucial role in chemotherapy-induced apoptosis and reducing cell viability. Moreover, decreasing m6A methyltransferase (e.g., methyltransferase-like 3, METTL3) expression through chemotherapeutic drug combinations reduced p53 mRNA m6A modification. p53 mRNA m6A modification blockage induced by S-adenosyl homocysteine or siRNA-mediated METTL3 inhibition enhanced HCC sensitivity to chemotherapy. Importantly, we observed that downregulation of METTL3 and upregulation of p53 expression by oral administration of chemotherapy drugs triggered apoptosis and xenograft tumor growth inhibition in nude mice. Based on these findings, we hypothesize that a METTL3-m6A-p53 axis could be a potential target in HCC therapy.
    Keywords:  Chemotherapy; Hepatocellular carcinoma; Target therapy; m6A; p53
    DOI:  https://doi.org/10.1007/s10495-022-01728-x
  8. Front Oncol. 2022 ;12 873020
      As one of the most common internal modifications in eukaryotic mRNA, N6-methyladenosine (m6A) modification is involved in the pathogenesis of many diseases, including hepatocellular carcinoma (HCC). In this study, we explored the prognostic significance of the expression of RNA binding motif protein 15B (RBM15B) in HCC, by studying specimens collected from clinical subjects. RBM15B is highly expressed in HCC patients and indicates a poor prognosis. Functionally, overexpression of RBM15B promotes HCC cell proliferation and invasion and induces sorafenib resistance in HCC cells. Mechanistically, we confirmed that RBM15B is transcriptionally activated by YY1 and regulates the stability of TRAM2 mRNA in an m6A-dependent manner. Overall, our results reveal a YY1-RBM15B-TRAM2 regulatory axis and highlight the critical role of RBM15B and m6A modifications in HCC. These findings may provide a novel mechanism and therapeutic targets for the treatment of HCC.
    Keywords:  N6-methyladenosine; RBM15B; TRAM2; hepatocellular carcinoma; mRNA stability
    DOI:  https://doi.org/10.3389/fonc.2022.873020
  9. BMC Med Genomics. 2022 May 05. 15(1): 103
       BACKGROUND: This is the first study to explore the potential functions and expression patterns of RNA N6-methyladenosine (m6A) and potential related genes in preeclampsia.
    METHODS: We identified two m6A modification patterns through unsupervised cluster analysis and validated them by principal component analysis. We quantified the relative abundance of specific infiltrating immunocytes using single-sample gene set enrichment analysis (ssGSEA) and the Wilcoxon test. To screen hub genes related to m6A regulators, we performed weighted gene coexpression network analysis. Functional enrichment analysis was conducted for differential signalling pathways and cellular processes. Preeclampsia patients were grouped by consensus clustering based on differentially expressed hub genes and the relationship between different gene-mediated classifications and clinical features.
    RESULTS: Two m6A clusters in preeclampsia, cluster A and cluster B, were determined based on the expression of 17 m6A modification regulators; ssGSEA revealed seven significantly different immune cell subtypes between the two clusters. A total of 1393 DEGs and nine potential m6A-modified hub genes were screened. We divided the patients into two groups based on the expression of these nine genes. We found that almost all the patients in m6A cluster A were classified into hub gene cluster 1 and that a lower gestational age may be associated with more m6A-associated events.
    CONCLUSIONS: This study revealed that hub gene-mediated classification is consistent with m6A modification clusters for predicting the clinical characteristics of patients with preeclampsia. Our results provide new insights into the molecular mechanisms of preeclampsia.
    Keywords:  Bioinformatic gene analysis; Biomarkers; Hub gene; N6-methyladenosine; Preeclampsia; Prognosis
    DOI:  https://doi.org/10.1186/s12920-022-01254-4
  10. Clin Exp Metastasis. 2022 May 07.
      N6-methyladenosine (m6A) is the most prevalent and internal modification that occurs in the messenger RNAs of eukaryotes. However, knowledge of the impact of these modifications on gene expression regulation remains limited. By using the in vitro MeRIP-seq and RNA-seq assays, we discovered that the mRNA demethylase FTO was significantly up-regulated in esophageal squamous cell carcinoma (ESCC) tissues and cells. Knockdown of FTO drastically suppressed the proliferation, migration, and invasion of ESCC cells. Furthermore, by using transcriptome-wide m6A-seq and RNA-seq assays, we identified ERBB2 is the target of FTO, which acts in concert in ESCC tumorigenesis and metastasis. Moreover, loss and gain functional studies suggested that the m6A reader YTHDF1 stabilizes ERBB2 mRNA via decoding the m6A modification. All these results uncovered a new signaling cascade, including FTO, YTHDF1, and ERBB2, which finely regulates the ESCC progression.
    Keywords:  ERBB2; Esophageal squamous cell carcinoma; FTO; N6-methyladenosine (m6A) modification; YTHDF1
    DOI:  https://doi.org/10.1007/s10585-022-10169-4
  11. Cell Death Discov. 2022 May 02. 8(1): 240
      Increasing evidence suggest the biological roles of N6-methyladenosine (m6A) and long noncoding RNAs (lncRNAs) in the bone disease, especially osteoarthritis (OA). However, the interaction of m6A and lncRNA in osteoarthritis is still unclear. Here, we found that a m6A-related lncRNA LINC00680 upregulated in the OA tissue and IL-1β-induced isolated primary chondrocytes. Functionally, in IL-1β-induced chondrocytes, silencing of LINC00680 recovered the proliferation and repressed the extracellular matrix (ECM) degradation. Mechanistically, m6A methyltransferase METTL3 combined tithe the m6A site of LINC00680 to up-regulate its expression. Moreover, LINC00680 interacted with SIRT1 mRNA through binding at m6A site on SIRT1 mRNA 3'-UTR, thereby enhancing the stability of SIRT1 mRNA. Overall, these findings exhibited a role of LINC00680/m6A/SIRT1 mRNA complex in chondrocytes. Taken together, the present study intends to uncover the mechanism by which METTL3-mediated LINC00680 accelerates OA progression, which may provide novel insight for OA.
    DOI:  https://doi.org/10.1038/s41420-022-00890-0
  12. Clin Transl Med. 2022 May;12(5): e778
       BACKGROUND: Solute carrier family 7 member 11 (SLC7A11) is overexpressed in multiple human tumours and functions as a transporter importing cystine for glutathione biosynthesis. It promotes tumour development in part by suppressing ferroptosis, a newly identified form of cell death that plays a pivotal role in the suppression of tumorigenesis. However, the role and underlying mechanisms of SLC7A11-mediated ferroptosis in hepatoblastoma (HB) remain largely unknown.
    METHODS: Reverse transcription quantitative real-time PCR (RT-qPCR) and western blotting were used to measure SLC7A11 levels. Cell proliferation, colony formation, lipid reactive oxygen species (ROS), MDA concentration, 4-HNE, GSH/GSSG ratio and cell death assays as well as subcutaneous xenograft experiments were used to elucidate the effects of SLC7A11 in HB cell proliferation and ferroptosis. Furthermore, MeRIP-qPCR, dual luciferase reporter, RNA pulldown, RNA immunoprecipitation (RIP) and RACE-PAT assays were performed to elucidate the underlying mechanism through which SLC7A11 was regulated by the m6A modification in HB.
    RESULTS: SLC7A11 expression was highly upregulated in HB. SLC7A11 upregulation promoted HB cell proliferation in vitro and in vivo, inhibiting HB cell ferroptosis. Mechanistically, SLC7A11 mRNA exhibited abnormal METTL3-mediated m6A modification, which enhanced its stability and expression. IGF2 mRNA-binding protein 1 (IGF2BP1) was identified as the m6A reader of SLC7A11, enhancing SLC7A11 mRNA stability and expression by inhibiting SLC7A11 mRNA deadenylation in an m6A-dependent manner. Moreover, IGF2BP1 was found to block BTG2/CCR4-NOT complex recruitment via competitively binding to PABPC1, thereby suppressing SLC7A11 mRNA deadenylation.
    CONCLUSIONS: Our findings demonstrated that the METTL3-mediated SLC7A11 m6A modification enhances HB ferroptosis resistance. The METTL3/IGF2BP1/m6A modification promotes SLC7A11 mRNA stability and upregulates its expression by inhibiting the deadenylation process. Our study highlights a critical role of the m6A modification in SLC7A11-mediated ferroptosis, providing a potential strategy for HB therapy through blockade of the m6A-SLC7A11 axis.
    Keywords:  IGF2BP1; SLC7A11; ferroptosis; hepatoblastoma; m6A methylation; resistance
    DOI:  https://doi.org/10.1002/ctm2.778
  13. Front Bioeng Biotechnol. 2022 ;10 846812
      Background: N6-methyladenosine (m6A) methylation played a key role in tumor growth. However, the relationship between m6A and soft tissue sarcoma (STS) was still unclear. Methods: The characterization and patterns of m6A modification in STS (TCGA-SARC and GSE17674) were analyzed comprehensively through bioinformatics and real-time polymerase chain reaction (RT-PCR). The effects of different m6A modification patterns on prognosis and immune infiltration of STS were further explored. Differentially expressed gene (DEG) analysis was performed. Moreover, an m6Ascore was constructed by principal component analysis (PCA). In addition, two immunotherapy datasets (IMvigor210 and GSE78220) and a sarcoma dataset (GSE17618) were used to evaluate the m6Ascore. Results: Huge differences were found in somatic mutation, CNV, and expression of 25 m6A regulators in STS. Two modification patterns (A and B) in STS were further identified and the m6A cluster A showed a better clinical outcome with a lower immune/stromal score compared with the m6A cluster B (p < 0.050).In addition to , most STS samples from m6A cluster A showed a high m6Ascore, which was related to mismatch repair and a better prognosis of STS (p < 0.001). In contrast, the m6A cluster B, characterized by a low m6Ascore, was related to the MYC signaling pathway, which led to a poor prognosis of STS. A high m6Ascore also contributed to a better outcome of PD-1/PD-L1 blockade immunotherapy. Conclusion: The modification patterns of 25 m6A regulators in the STS microenvironment were explored comprehensively. The novel m6Ascore effectively predicted the characteristics of the tumor microenvironment (TME) and outcome in STS and provided novel insights for future immunotherapy.
    Keywords:  immunotherapy; m6A; soft tissue sarcoma; survival; tumor microenvironment
    DOI:  https://doi.org/10.3389/fbioe.2022.846812
  14. J Transl Med. 2022 May 04. 20(1): 197
       BACKGROUND: N6-methyladenosine (m6A) RNA methylation plays a critical role in key genetic events for various cancers; yet, how m6A functions within the tumor microenvironment (TME) remains to be elucidated.
    METHODS: A total of 65,362 single cells from single-cell RNA-seq data derived from 33 CRC tumor samples were analyzed by nonnegative matrix factorization (NMF) for 23 m6A RNA methylation regulators. CRC and Immunotherapy cohorts from public repository were used to determine the prognosis and immune response of TME clusters.
    RESULTS: The fibroblasts, macrophages, T and B cells were respectively grouped into 4 to 5 subclusters and then classified according to various biological processes and different marker genes. Furthermore, it revealed that the m6A RNA methylation regulators might be significantly related to the clinical and biological features of CRC, as well as the pseudotime trajectories of main TME cell types. Bulk-seq analysis suggested that these m6A-mediated TME cell subclusters had significant prognostic value for CRC patients and distinguished immune response for patients who underwent ICB therapy, especially for the CAFs and macrophages. Notably, CellChat analysis revealed that RNA m6A methylation-associated cell subtypes of TME cells manifested diverse and extensive interaction with tumor epithelial cells. Further analysis showed that ligand-receptor pairs, including MIF -  (CD74 + CXCR4), MIF -  (CD74 + CD44), MDK-NCL and LGALS9 - CD45, etc. mediated the communication between m6A associated subtypes of TME cells and tumor epithelial cells.
    CONCLUSIONS: Taken together, our study firstly revealed the m6A methylation mediated intercellular communication of the tumor microenvironment in the regulation of tumor growth and antitumor immunomodulatory processes.
    Keywords:  Colorectal cancer; Immunotherapy; Prognosis; Single-cell; Tumor microenvironment; m6A
    DOI:  https://doi.org/10.1186/s12967-022-03395-7
  15. Front Genet. 2022 ;13 869950
      N6-methyladenosine (m6A) is the most common and conserved internal eukaryotic mRNA modification. m6A modification is a dynamic and reversible post-transcriptional regulatory modification, initiated by methylase and removed by RNA demethylase. m6A-binding proteins recognise the m6A modification to regulate gene expression. Recent studies have shown that altered m6A levels and abnormal regulator expression are crucial in the ageing process and the occurrence of age-related diseases. In this review, we summarise some key findings in the field of m6A modification in the ageing process and age-related diseases, including cell senescence, autophagy, inflammation, oxidative stress, DNA damage, tumours, neurodegenerative diseases, diabetes, and cardiovascular diseases (CVDs). We focused on the biological function and potential molecular mechanisms of m6A RNA methylation in ageing and age-related disease progression. We believe that m6A modification may provide a new target for anti-ageing therapies.
    Keywords:  N6-methyladenosine; RNA methylation; aging; aging-related disease; epigenetics
    DOI:  https://doi.org/10.3389/fgene.2022.869950
  16. Cell Death Discov. 2022 May 02. 8(1): 237
      N6-methyladenosine (m6A) is a key area in Epigenetics and has been increasingly focused these years. In the m6A process, readers recognize the m6A modification on mRNAs or noncoding RNAs and mediate different downstream events. Emerging studies have shown that YTHDC1, an important m6A reader, plays a key role in many biological functions and disease progression, especially cancers. Here we summarized the current mechanisms of YTHDC1 in biological functions and diseases and offered guidance for future researches to provide potential strategy for clinical diagnose and therapy.
    DOI:  https://doi.org/10.1038/s41420-022-01040-2
  17. Comput Struct Biotechnol J. 2022 ;20 1785-1797
      The cGAS-STING signaling plays pivotal roles not only in host antiviral defense but also in various noninfectious contexts. Compared with protein-coding genes, much less was known about long noncoding RNAs involved in this pathway. Here, we performed an integrative study to elucidate the lncRNA repertoire and the mechanisms modulating lncRNA's expression following cGAS-STING signaling activation. We uncovered a reliable set of 672 lncRNAs closely linked to cGAS-STING signaling activation (cs-lncRNA), which might be associated with type-I interferon response and infection-related phenotypes. The ChIP-seq analysis demonstrated that cs-lncRNA was strongly regulated at the transcriptional level. We further found N6-methyladenosine (m6A) regulatory machinery was indispensable for establishing cs-lncRNA repertoire via modulating m6A modification on cs-lncRNA transcripts and promoting the expression of signaling transduction key components, including IFNAR1. Loss of IFNAR1 led to the dysregulation of cs-lncRNAs resembled that of loss of an essential subunit of m6A writer METTL14. We also found m6A system affected transcriptional machinery to modulate cs-lncRNAs by targeting multiple crucial transcription factors. Inhibiting an m6A modification regulated transcription factor, EZH2, markedly enhanced the expression pattern of cs-lncRNAs. Taken together, our results uncovered the composition of the cs-lncRNAs and revealed m6A-mediated modulation coupled with transcriptional regulation significantly shaped cs-lncRNA repertoire.
    Keywords:  N6-methyladenosine (m6A); Transcription factor; Transcriptional regulation; cGAS-STING signaling; lncRNA
    DOI:  https://doi.org/10.1016/j.csbj.2022.04.002
  18. Science. 2022 May 05. eabe9582
      N6-methyladenosine (m6A) is the most abundant internal modification on mammalian messenger RNA (mRNA). It is installed by a writer complex and can be reversed by erasers such as the fat mass and obesity-associated protein (FTO). Despite extensive research, the primary physiological substrates of FTO in mammalian tissues and development remain elusive. Here, we show that FTO mediates m6A demethylation of long-interspersed element-1 (LINE1) RNA in mouse embryonic stem cells (mESCs), regulating LINE1 RNA abundance and the local chromatin state, which in turn modulates transcription of LINE1-containing genes. FTO-mediated LINE1 RNA m6A demethylation also plays regulatory roles in shaping chromatin state and gene expression during mouse oocyte and embryonic development. Our results suggest broad effects of LINE1 RNA m6A demethylation by FTO in mammals.
    DOI:  https://doi.org/10.1126/science.abe9582
  19. Mol Ther Nucleic Acids. 2022 Jun 14. 28 464-476
      RNA chemical modifications are a new but rapidly developing field. They can directly affect RNA splicing, transport, stability, and translation. Consequently, they are involved in the occurrence and development of diseases that have been studied extensively in recent years. However, few studies have focused on the correlation between chemical modifications and drug effects. Here, we provide a landscape of six RNA modifications in pharmacogene RNA (pharmacoepitranscriptomics) to fully clarify the correlation between chemical modifications and drugs. We performed systematic and comprehensive analyses on pharmacoepitranscriptomics, including basic characteristics of RNA modification and modification-associated mutations and drugs affected by them. Our results show that chemical modifications are common in pharmacogenes, especially N6-methyladenosine (m6A) modification. In addition, we found a very close relationship between chemical modifications and anti-tumor drugs. More interestingly, the results demonstrate the importance of m6A modification for anti-tumor drugs, especially for drugs in triple-negative breast cancer (TNBC), ovarian cancer, and acute myelocytic leukemia (AML). These results indicate that pharmacoepitranscriptomics could be a new source of drug-effect biomarkers, especially for m6A and anti-tumor drugs.
    Keywords:  MT: bioinformatics; N6-methyladenosine; anti-tumor drugs; cancer; chemical modifications; drug effects; m6A; pharmacoepitranscriptomics; pharmacogene
    DOI:  https://doi.org/10.1016/j.omtn.2022.04.001
  20. J Cancer. 2022 ;13(7): 2105-2125
      Background: Neuroblastoma (NB) is a pediatric cancer occurring in the peripheral nervous system. A demethylase, alkylation repair homolog protein 5 (ALKBH5), is one type of N6-methyladenosine (m6A) eraser that plays a tumor-suppressive role in a variety of cancers. The significance of carbohydrate metabolism in cancer has been intensively investigated over the years, but the correlation between ALKBH5 and glucose metabolism in NB remains to be elucidated. Methods: Based on the overlapped genes (DE-GRGs) of ALKBH5-related differentially expressed genes (ALKBH5-DEGs) in GSE62564 (n=498) and genes related to glucose metabolism (GRGs), a LASSO regression model was constructed. External validations with datasets (EGAS00001001308, n=139 & GSE16476, n=88) and the NB samples from Shanghai Children's Hospital (SCH) were performed. Meanwhile, biological and clinical utility, immune cell subtypes and drug sensitivity were assessed. Results: ALKBH5 was significantly correlated with better overall survival (OS) in NB patients, and gene set enrichment analysis (GSEA) showed its enrichment in GO/ KEGG terms regarding glucose metabolism. 27 of the 31 DE-GRGs were included in the LASSO screen after the univariate analysis. A prognostic glucometabolic model including AHCY, NCAN, FBP2, GALNT3 and AKR1C2 was established with the internal and external validation with biological experiments: the high-risk subtype compared to the low-risk subtype showed oncogenic and MYCN-related malignancy, glucometabolic dysregulation, poor prognosis and immunosuppression. TGX-221 was predicted to be a potential therapeutic drug and validated to suppress NB oncogenes including MYCN, AHCY and NCAN and immunosuppressive DNMT1 in NB cells. Conclusion: ALKBH5 was closely related to glucometabolic processes, and our prognostic model had high application value in predicting & assessing the OS of NB patients, and even served potential drug targets.
    Keywords:  ALKBH5; drug targets.; glucometabolic genes; m6A eraser; neuroblastoma; prognostic model
    DOI:  https://doi.org/10.7150/jca.69408
  21. Am J Physiol Cell Physiol. 2022 May 04.
      Fibroblasts play an important role in the pathogenic mechanisms of several socially significant diseases, including pulmonary and cardiovascular fibrosis, liver cirrhosis, systemic sclerosis, progressive kidney disease. The alterations of the epitranscriptome, including more than 170 distinct post-transcriptional RNA modifications or editing events, justified their investigation as an important modulator of fibrosis. Recent development of high-throughput methods allows the identification of RNA modification sites and their mechanistic aspect in the fibrosis development. The most common RNA modification is methylation of N6-adenosine deposited by the m6A methyltransferase complex (METTL3/14/16, WTAP, KIAA1429, and RBM15/15B), erased by demethylases (FTO and ALKBH5), and recognized by binding proteins (e.g., YTHDF1/2/3, YTHDC1/2, IGF2BP1/2/3, etc.). Adenosine to inosine (A-to-I) RNA editing is another abundant editing event converting adenosine to inosine in double-stranded RNA regions through the action of the adenosine deaminase (ADAR) proteins. Last, but not least, 5-methylcytosine (m5C) regulates the stability and translation of mRNAs. All those RNA modifications have been observed in mRNA as well as the non-coding regions of pre-mRNA and ncRNAs, and demonstrate to be involved in fibrosis in different cellular and animal models. This Mini-Review focuses on the latest research on epitranscriptomic marks related to fibroblast biology and fibrosis as well as elucidates the future research directions in this context.
    Keywords:  RNA editing; RNA methylation; epitranscriptomics; fibroblast; fibrosis
    DOI:  https://doi.org/10.1152/ajpcell.00121.2022
  22. Mol Cancer. 2022 May 06. 21(1): 109
       BACKGROUND: Emerging evidence suggest the critical role of circular RNAs (circRNAs) in disease development especially in various cancers. However, the oncogenic role of circRNAs in hepatocellular carcinoma (HCC) is still largely unknown.
    METHODS: RNA sequencing was performed to identify significantly upregulated circRNAs in paired HCC tissues and non-tumor tissues. CCK-8 assay, colony formation, transwell, and xenograft mouse models were used to investigate the role of circRNAs in HCC proliferation and metastasis. Small interfering RNA (siRNA) was used to silence gene expression. RNA immunoprecipitation, biotin pull-down, RNA pull-down, luciferase reporter assay and western blot were used to explore the underlying molecular mechanisms.
    RESULTS: Hsa_circ_0095868, derived from exon 5 of the MDK gene (named circMDK), was identified as a new oncogenic circRNA that was significantly upregulated in HCC. The upregulation of circMDK was associated with the modification of N6-methyladenosine (m6A) and poor survival in HCC patients. Mechanistically, circMDK sponged miR-346 and miR-874-3p to upregulate ATG16L1 (Autophagy Related 16 Like 1), resulting to the activation of PI3K/AKT/mTOR signaling pathway to promote cell proliferation, migration and invasion. Poly (β-amino esters) (PAEs) were synthesized to assist the delivery of circMDK siRNA (PAE-siRNA), which effectively inhibited tumor progression without obvious adverse effects in four liver tumor models including subcutaneous, metastatic, orthotopic and patient-derived xenograft (PDX) models.
    CONCLUSIONS: CircMDK could serve as a potential tumor biomarker that promotes the progression of HCC via the miR-346/874-3p-ATG16L1 axis. The PAE-based delivery of siRNA improved the stability and efficiency of siRNA targeting circMDK. The PAE-siRNA nanoparticles effectively inhibited HCC proliferation and metastasis in vivo. Our current findings offer a promising nanotherapeutic strategy for the treatment of HCC.
    Keywords:  ATG16L1; Apoptosis; Hepatocellular carcinoma (HCC); IGF2BP1; N6-methyladenosine (m6A); Nanoparticles (NPs); Poly (β-amino esters) (PAEs); circRNA
    DOI:  https://doi.org/10.1186/s12943-022-01575-z
  23. J Transl Med. 2022 May 04. 20(1): 198
       BACKGROUND: Serine/arginine-rich splicing factor 9 (SRSF9) is a classical RNA-binding protein that is essential for regulating gene expression programs through its interaction with target RNA. Whether SRSF9 plays an essential role in colorectal cancer (CRC) progression and can serve as a therapeutic target is largely unknown. Here, we highlight new findings on the role of SRSF9 in CRC progression and elucidate the underlying mechanism.
    METHODS: CRC cell lines and clinical tissue samples were used. qRT-PCR, Western blotting, immunohistochemistry (IHC), gain- and loss-of-function assays, animal xenograft model studies, bioinformatic analysis, methylated single-stranded RNA affinity assays, gene-specific m6A quantitative qRT-PCR, dual-luciferase reporter assays and RNA stability assays were performed in this study.
    RESULTS: The expression level of SRSF9 was higher in CRC cell lines than that in an immortal human intestinal epithelial cell line. Overexpression of SRSF9 was positively associated with lymph node metastasis and Dukes stage. Functionally, SRSF9 promoted cell proliferation, migration and invasion in vitro and xenograft growth. The results of bioinformatic analysis indicated that DSN1 was the downstream target of SRSF9. In CRC cells and clinical tissue samples, the expression of SRSF9 was positively associated with the expression of DSN1. Knockdown of DSN1 partially inhibited the SRSF9-induced phenotype in CRC cells. Mechanistically, we further found that SRSF9 is an m6A-binding protein and that m6A modifications were enriched in DSN1 mRNA in CRC cells. Two m6A modification sites (chr20:36773619-36773620 and chr20:36773645-chr20:36773646) in the SRSF9-binding region (chr20:36773597-36773736) of DSN1 mRNA were identified. SRSF9 binds to DSN1 in an m6A motif- and dose-dependent manner. SRSF9 modulates the expression of DSN1 in CRC cells. Such expression regulation was largely impaired upon methyltransferase METTL3 knockdown. Moreover, knockdown of SRSF9 accelerated DSN1 mRNA turnover, while overexpression of SRSF9 stabilized DSN1 mRNA in CRC cells. Such stabilizing was also weakened upon METTL3 knockdown.
    CONCLUSION: Overexpression of SRSF9 was associated with lymph node metastasis and Dukes stage in CRC. Knockdown of DSN1 eliminated the effects by SRSF9 overexpression in CRC. Our results indicated that SRSF9 functions as an m6A-binding protein (termed "reader") by enhancing the stability of DSN1 mRNA in m6A-related manner. Our study is the first to report that SRSF9-mediated m6A recognition has a crucial role in CRC progression, and highlights SRSF9 as a potential therapeutic target for CRC management.
    Keywords:  Colorectal cancer; Component of MIS12 kinetochore complex; DSN1; Epigenetics; N6-Methyladenosine (m6A); SRSF9; Serine and arginine rich splicing factor 9
    DOI:  https://doi.org/10.1186/s12967-022-03399-3
  24. Mol Cell. 2022 Apr 27. pii: S1097-2765(22)00321-5. [Epub ahead of print]
      The p53 transcription factor drives anti-proliferative gene expression programs in response to diverse stressors, including DNA damage and oncogenic signaling. Here, we seek to uncover new mechanisms through which p53 regulates gene expression using tandem affinity purification/mass spectrometry to identify p53-interacting proteins. This approach identified METTL3, an m6A RNA-methyltransferase complex (MTC) constituent, as a p53 interactor. We find that METTL3 promotes p53 protein stabilization and target gene expression in response to DNA damage and oncogenic signals, by both catalytic activity-dependent and independent mechanisms. METTL3 also enhances p53 tumor suppressor activity in in vivo mouse cancer models and human cancer cells. Notably, METTL3 only promotes tumor suppression in the context of intact p53. Analysis of human cancer genome data further supports the notion that the MTC reinforces p53 function in human cancer. Together, these studies reveal a fundamental role for METTL3 in amplifying p53 signaling in response to cellular stress.
    Keywords:  DNA damage; METTL14; METTL3; N(6)-methyladenosine (m6A) modification; epitranscriptomics; lung cancer; mass spectrometry; methyltransferase complex; p53; tumor suppressor
    DOI:  https://doi.org/10.1016/j.molcel.2022.04.010
  25. Clin Transl Med. 2022 May;12(5): e738
       BACKGROUND: Dysregulation of the epitranscriptome causes abnormal expression of oncogenes in the tumorigenic process. Previous studies have shown that NAT10 can regulate mRNA translation efficiency through RNA acetylation. However, the role of NAT10-mediated acetylation modification in bladder cancer remains elusive.
    METHODS: The clinical value of NAT10 was estimated according to NAT10 expression pattern based on TCGA data set and the tumor tissue array. Acetylated RNA immunoprecipitation sequencing was utilized to explore the role of NAT10 in mRNA ac4C modification. Translation efficiency and mRNA stability assay were applied to study the effect of NAT10-deletion on target genes. The nude mouse model and genetically engineered mice were conducted to further verify the characteristics of NAT10 in promoting BLCA progression and regulating downstream targets.
    RESULTS: NAT10 was essential for the proliferation, migration, invasion, survival and the stem-cell-like properties of bladder cancer cell lines. NAT10 was responsible for mRNA ac4C modification in BLCA cells, including BCL9L, SOX4 and AKT1. Deficient NAT10 in both xenograft and transgenic mouse models of bladder cancer reduced the tumor burden. Furthermore, acetylated RNA immunoprecipitation sequencing data and RNA immunoprecipitation qPCR results revealed that NAT10 is responsible for a set of ac4C mRNA modifications in bladder cancer cells. Inhibition of NAT10 led to a loss of ac4C peaks in these transcripts and represses the mRNA's stability and protein expression. Mechanistically, the ac4C reduction modification in specific regions of mRNAs resulting from NAT10 downregulation impaired the translation efficiency of BCL9L, SOX4 and AKT1 as well as the stability of BCL9L, SOX4.
    CONCLUSIONS: In summary, these findings provide new insights into the dynamic characteristics of mRNA's post-transcriptional modification via NAT10-dependent acetylation and predict a role for NAT10 as a therapeutic target in bladder cancer.
    HIGHLIGHTS: NAT10 is highly expressed in BLCA patients and its abnormal level predicts bladder cancer progression and low overall survival rate. NAT10 is necessary and sufficient for BLCA tumourigenic properties. NAT10 is responsible for ac4C modification of target transcripts, including BCL9L, SOX4 and AKT1. NAT10 may serve as an effective and novel therapeutic target for BLCA.
    Keywords:  N4-acetylcytidine; NAT10; bladder cancer; mRNA
    DOI:  https://doi.org/10.1002/ctm2.738
  26. Oncogene. 2022 Apr 30.
      Thermal ablation is a main curative therapy for early-stage hepatocellular carcinoma (HCC). However, insufficient ablation has been shown to promote HCC progression. E3 ligases have been approved to play important roles in malignant tumors. Whether E3 ligases are involved in HCC progression caused by insufficient ablation remains unclear. Herein, using RNA-sequencing coupled with an in vitro loss-of-function screen, we found that the E3 ligase Neuronal Precursor cell-expressed Developmentally Downregulated 4 (Nedd4) was upregulated in HCC insufficient ablation tissues and promoted HCC cells migration. The upregulation of Nedd4 was induced by METTL14-mediated N6-methyladenosine modification after sublethal heat treatment. Knockdown of Nedd4 inhibited HCC metastasis and growth in vitro and in vivo. Mechanistically, Nedd4 enhanced TGF-β signal transduction mediated tumor progression by directly binding to TGF-β type I receptor (TGFBR1) and forming K27-linked ubiquitin at Lysine 391. Additionally, the adverse effect on HCC of sublethal heat treatment was mediated by Nedd4. Clinically, high Nedd4 expression was positively correlated with aggressive tumor phenotypes and poor prognosis in HCC patients. Patient-derived xenograft (PDX) model confirmed this conclusion. Collectively, this study demonstrated that Nedd4 induced by insufficient ablation plays a crucial role in promoting HCC progression and provides a novel therapeutic target for HCC.
    DOI:  https://doi.org/10.1038/s41388-022-02334-6
  27. Front Vet Sci. 2022 ;9 757115
      N6-methyladenine (m6A) RNA undergoes epigenetic modification, which is the most extensive intermediate chemical modification in mRNA. Although this modification occurs in all living organisms, it is the most widely studied among mammals. However, to date, no study has investigated the m6A transcriptome-wide map of yak and its potential biological functions in muscle development. In this study, the differences of m6A methylation and gene expression in yak muscle development belonging to three age groups, namely 3 years (group A), 6 months (group M), and 90-day-old fetuses (group E), were determined by using methylated RNA immunoprecipitation sequencing (MeRIP-Seq) and RNA sequencing (RNA-Seq). In these three groups, a total of 6,278 (A), 9,298 (E), and 9,584 (M) m6A peaks were identified, with average densities between 1.02 and 2.01. m6A peaks were mostly enriched in the stop codon, 3' untranslated region (UTR) region, and inner long exon region with consensus motifs of UGACA. In all the three stages, the m6A peak enrichment level was significantly negatively correlated with mRNA abundance (Pearson's correlation coefficient r = -0.22 to -0.32, p < 10-16). The functional enrichment of genes consistently modified by m6A methylation, particularly those genes that regulate cell differentiation as well as muscle growth and development, was observed at all three stages. Moreover, m6A abundance was negatively associated with gene expression levels, indicating that m6A might play a vital role in modulating gene expression during yak muscle development. This comprehensive map thus provides a solid foundation for determining the potential functional role of m6A RNA modification in yak muscle growth.
    Keywords:  MeRIP-Seq; longissimus dorsi; muscle development; transcriptional regulation; yak
    DOI:  https://doi.org/10.3389/fvets.2022.757115
  28. Pathol Res Pract. 2022 Apr 28. pii: S0344-0338(22)00163-7. [Epub ahead of print]234 153919
      Numerous studies show that some biomarkers are aberrantly expressed in endometrial endometrioid adenocarcinoma (EMAC) and endometrial atypical hyperplasia/endometrioid intraepithelial neoplasia (EAH/EIN) compared to endometrial benign lesions. Because of low sensitivity and/or specificity, the utility of these markers to distinguish EMAC and EAH/EIN from benign endometrial lesions is limited. YTH domain family 2 (YTHDF2) is a functional N6-methyladenosine (m6A)-specific reader protein that mainly regulates mRNA stability. Aberrant YTHDF2 expression has been reported in many cancers and plays important functions in tumorigenesis and cancer progression. However, its expression in endometrial benign and malignant lesions has not been investigated. We evaluated YTHDF2 mRNA and protein expression in EMAC and normal endometrium using the UALCAN database and validated the bioinformatic results in EMAC cells using qRT-PCR, Western blot, and immunohistochemical (IHC) staining. We found that YTHDF2 was weakly expressed in normal endometrium, benign endometrial lesions, endometrial hyperplasia without atypia, and adenomyosis. In contrast, YTHDF2 was upregulated in EAH/EIN and EMAC. These results indicate that YTHDF2 immunostaining may be a useful tool to distinguish EAH/EIN from EHWA. Finally, YTHDF2 expression can accurately assess the depth of myometrial invasion (DMI) in EMAC when EMAC coexists with adenomyosis.
    Keywords:  Adenomyosis; Endometrial atypical hyperplasia; Endometrioid adenocarcinoma; Endometrioid intraepithelial neoplasia; YTHDF2
    DOI:  https://doi.org/10.1016/j.prp.2022.153919