bims-rimeca Biomed News
on RNA methylation in cancer
Issue of 2021‒08‒08
twenty-four papers selected by
Sk Ramiz Islam
Saha Institute of Nuclear Physics

  1. Front Oncol. 2021 ;11 702983
      Acquired chemoresistance is a major limiting factor in the clinical treatment of glioblastoma (GBM). However, the mechanism by which GBM acquires therapeutic resistance remains unclear. Here, we aimed to investigate whether METTL3-mediated N6-methyladenosine (m6A) modification contributes to the temozolomide (TMZ) resistance in GBM. We demonstrated that METTL3 METTL3-mediated m6A modification were significantly elevated in TMZ-resistant GBM cells. Functionally, METTL3 overexpression impaired the TMZ-sensitivity of GBM cells. In contrast, METTL3 silencing or DAA-mediated total methylation inhibition improved the sensitivity of TMZ-resistant GBM cells to TMZ in vitro and in vivo. Furthermore, we found that two critical DNA repair genes (MGMT and APNG) were m6A-modified by METTL3, whereas inhibited by METTL3 silencing or DAA-mediated total methylation inhibition, which is crucial for METTL3-improved TMZ resistance in GBM cells. Collectively, METTL3 acts as a critical promoter of TMZ resistance in glioma and extends the current understanding of m6A related signaling, thereby providing new insights into the field of glioma treatment.
    Keywords:  METTL3; N6-methyladenosine (m 6 A); glioblastoma; resistance; temozolomide
  2. Front Cell Dev Biol. 2021 ;9 617172
      The N6-methyladenosine (m6A) RNA modification regulates the expression of genes associated with various biological and pathological processes, including spontaneous abortion (SA). The aim of this study was to determine the role of the m6A demethylase fat mass and obesity (FTO)- associated protein in SA. The FTO,IGF2BP1 and IGF2BP2 mRNA levels were significantly lower in the chorionic villi obtained from spontaneously aborted pregnancies compared to that of normal pregnancies, while the expression levels of METTL3 and WTAP were significantly elevated. However, ALKBH5, YTHDF2, and IGF2BP3 were elevated with no statistical significance between groups. In addition, MDA was elevated and SOD levels were decreased in the villi tissues of the SA group compared to the normal group, which was indicative of placental oxidative stress in the former. Furthermore, the expression of FTO and HLA-G were significantly decreased in the trophoblasts of the SA patients compared to that of normal pregnant women, while that of m6A was markedly higher in the former. In addition, the HLA-G and VEGFR mRNA levels were downregulated in the SA versus the control group, and that of MMP2, MMP7, MMP9 and VEGFA were upregulated. Finally, The RIP assay showed significantly decreased levels of FTO-bound HLA-G, VEGFR and MMP9 RNA in SA patients (P < 0.05), which corresponded to an increase in transcripts enriched with the m6A antibody (P < 0.05). However, compared with normal pregnant women, the levels of HLA-G, VEGFA, VEGFR, and MMP2 mRNA bound by YTHDF2 were significantly decreased in SA patients. Compared to the normal pregnant women, both FTO- and m6A-bound MMP7 were significantly increased in SA patients (P < 0.05), but YTHDF2 almost unbound to MMP7 mRNA. In summary, the downregulation of FTO in the chorionic villi disrupts immune tolerance and angiogenesis at the maternal-fetal interface, resulting in aberrant methylation and oxidative stress that eventually leads to SA.
    Keywords:  FTO; RNA methylation; m6A; spontaneous abortion; villous
  3. Theranostics. 2021 ;11(16): 7640-7657
      Background: Since primary prostate cancer (PCa) can advance to the life-threatening metastatic PCa, exploring the molecular mechanisms underlying PCa metastasis is crucial for developing the novel targeted preventive strategies for decreasing the mortality of PCa. RNA N6-methyladenosine (m6A) is an emerging regulatory mechanism for gene expression and its specific roles in PCa progression remains elusive. Methods: Western blotting, quantitative real-time PCR and immunohistochemical analyses were used to detect target gene expression in PCa cells in vitro and prostate tissues from patients. RNA immunoprecipitation was conducted to analyze the specific binding of mRNA to the target protein. Migration and invasion assays were used to assess the migratory capacities of cancer cells. The correlation between target gene expression and survival rate of PCa patients was analyzed based the TCGA database. Results: We found that total RNA N6-methyladenosine (m6A) modification levels were markedly upregulated in human PCa tissues due to increased expression of methyltransferase like 3 (METTL3). Further studies revealed that the migratory and invasive capacities of PCa cells were markedly suppressed upon METTL3 knockdown. Mechanistically, METTL3 mediates m6A modification of USP4 mRNA at A2696, and m6A reader protein YTHDF2 binds to and induces degradation of USP4 mRNA by recruiting RNA-binding protein HNRNPD to the mRNA. Decrease of USP4 fails to remove the ubiquitin group from ELAVL1 protein, resulting in a reduction of ELAVL1 protein. Lastly, downregulation of ELAVL1 in turn increases ARHGDIA expression, promoting migration and invasion of PCa cells. Conclusions: Our findings highlight the role of METTL3 in modulating invasion and metastasis of PCa cells, providing insight into promising therapeutic strategies for hindering PCa progressing to deadly metastases.
    Keywords:  METTL3; m6A; metastasis; prostate cancer; therapeutic strategies
  4. BMC Cancer. 2021 Jul 31. 21(1): 876
      BACKGROUND: m6A is the most prevalent and abundant form of mRNA modifications and is closely related to tumor proliferation, differentiation, and tumorigenesis. In this study, we try to conduct an effective prediction model to investigated the function of m6A RNA methylation modulators in pancreatic adenocarcinoma and estimated the potential association between m6A RNA methylation modulators and tumor microenvironment infiltration for optimization of treatment.METHODS: Expression of 28 m6A RNA methylation modulators and clinical data of patients with pancreatic adenocarcinoma and normal samples were obtained from TCGA and GTEx database. Differences in the expression of 28 m6A RNA methylation modulators between tumour (n = 40) and healthy (n = 167) samples were compared by Wilcoxon test. LASSO Cox regression was used to select m6A RNA methylation modulators to analyze the relationship between expression and clinical characteristics by univariate and multivariate regression. A risk score prognosis model was conducted based on the expression of select m6A RNA methylation modulators. Bioinformatics analysis was used to explore the association between the m6Ascore and the composition of infiltrating immune cells between high and low m6Ascore group by CIBERSORT algorithm. Evaluation of m6Ascore for immunotherapy was analyzed via the IPS and three immunotherapy cohort. Besides, the biological signaling pathways of the m6A RNA methylation modulators were examined by gene set enrichment analysis (GSEA).
    RESULTS: Expression of 28 m6A RNA methylation modulators were upregulated in patients with PAAD except for MTEEL3. An m6Ascore prognosis model was established, including KIAA1429, IGF2BP2, IGF2BP3, METTL3, EIF3H and LRPPRC was used to predict the prognosis of patients with PAAD, the high risk score was an independent prognostic indicator for pancreatic adenocarcinoma, and a high risk score presented a lower overall survival. In addition, m6Ascore was related with the immune cell infiltration of PAAD. Patients with a high m6Ascore had lower infiltration of Tregs and CD8+T cells but a higher resting CD4+ T infiltration. Patients with a low m6Ascore displayed a low abundance of PD-1, CTLA-4 and TIGIT, however, the IPS showed no difference between the two groups. The m6Ascore applied in three immunotherapy cohort (GSE78220, TCGA-SKCM, and IMvigor210) did not exhibit a good prediction for estimating the patients' response to immunotherapy, so it may need more researches to figure out whether the m6A modulator prognosis model would benefit the prediction of pancreatic patients' response to immunotherapy.
    CONCLUSION: Modulators involved in m6A RNA methylation were associated with the development of pancreatic cancer. An m6Ascore based on the expression of IGF2BP2, IGF2BP3, KIAA1429, METTL3, EIF3H and LRPPRC is proposed as an indicator of TME status and is instrumental in predicting the prognosis of pancreatic cancer patients.
    Keywords:  Immune cell infiltration; Immunotherapy; Pancreatic adenocarcinoma; Prognostic; m6A RNA methylation modulators; m6Ascore
  5. Oncogene. 2021 Aug 06.
      N6-methyladenosine (m6A) is the most prevalent RNA epigenetic regulator in cancer. However, the understanding of m6A modification on lipid metabolism regulation in colorectal cancer (CRC) is very limited. Here, we observed that human CRCs exhibited increased m6A mRNA methylation mediated by dysregulation of m6A erasers and readers. By performing methylated RNA-immunoprecipitation sequencing (MeRIP-seq) and transcriptomic sequencing (RNA-seq), we identified DEGS2 as a downstream target of m6A dysregulation. Overexpression or knockdown of DEGS2 confirmed the role of DEGS2 in proliferation, invasion and metastasis of CRC both in vitro and in vivo. Mechanistic studies identified the specific m6A modification site within DEGS2 mRNA, and mutation of this target site was found to drastically enhance the proliferative and invasive ability of CRC cells in vitro and promote tumorigenicity in vivo. Lipidome analysis showed that lipid metabolism was dysregulated in CRC. Moreover, ceramide synthesis was suppressed due to DEGS2 upregulation mediated by m6A modification in CRC tissues. Our findings highlight that the function of DEGS2 m6A methylation in CRC and extend the understanding of the importance of RNA epigenetics in cancer biology.
  6. Cancer Lett. 2021 Jul 30. pii: S0304-3835(21)00381-5. [Epub ahead of print]518 256-265
      N6-methyladenosine (m6A), the most prevalent internal modification in eukaryotic mRNAs, regulates gene expression at the post-transcriptional level. The reader proteins of m6A, mainly YTH domain-containing proteins, specifically recognize m6A-modified mRNAs and regulate their metabolism. Recent studies have highlighted essential roles of m6A readers in the initiation and development of human cancers. In this review, we summarize recent findings about the biological functions of YTH domain proteins in cancers, the underlying mechanisms, and clinical implications. Gene expression reprogramming by dysregulated m6A reader proteins offers potential targets for cancer treatment, while targeted m6A editors and readers provide tools to manipulate m6A metabolism in cancers.
    Keywords:  Cancer; YTH domain protein; m(6)A editor; m(6)A modification; mRNA metabolism
  7. Am J Cancer Res. 2021 ;11(7): 3688-3697
      Genetic alterations in N6-methyladenosine (m6A) regulatory genes are observed in many cancers. Recent studies have shown that newly identified m6A regulatory gene family (IGF2BPs; IGF2BP1, IGF2BP2, and IGF2BP3) were highly expressed in various types of cancer that stabilize and promote translation of multiple oncogenes, resulting in tumor development, survival and drug resistance. However, the oncogenic roles and prognostic values of IGF2BPs in head and neck squamous cell carcinoma (HNSCC) remain largely unknown. In this study, we examined the m6A regulatory genes alteration, their mRNAs expression and the prognostic values in HNSCC. We also analyzed the interaction network and functional enrichment of m6A regulators. Our results showed that m6A regulatory genes were altered in 41% (205/504) of HNSCC patients, of which IGF2BP2 was amplified in 20% (101/504) of HNSCC patents and positively correlated with its mRNA expression. Importantly, we have validated the expression of IGF2BP2 in HNSCC and normal tissue samples. Interestingly, we also found that the IGF2BP2 was frequently co-amplified with the most common oncogenes in HNSCC patients. In addition, this study found that other m6A regulatory genes such as METTL3, METTL14, WTAP, KIAA1429, ZC3H13, RBM15, ALKBH5, FTO, YTHDF1, YTHDF2, YTHDF3, YTHDC1, IGF2BP1, and IGF2BP3 were significantly upregulated in HNSCC samples. Moreover, patients with high expression of IGF2BP1, IGF2BP2, and IGF2BP3 had poor overall survival (OS) than those with low expression. Therefore, it is evident that IGF2BP family plays a key role in the oncogenesis of HNSCC and might serve as novel prognostic biomarkers and potential therapeutic targets in HNSCC.
    Keywords:  HNSCC; m6A regulators; oncogenes; poor prognosis; tumorigenesis
  8. Int J Biol Sci. 2021 ;17(11): 2718-2736
      Cancer stemness, mainly consisting of chemo-resistance, radio-resistance, tumorigenesis, metastasis, tumor self-renewal, cancer metabolism reprogramming, and tumor immuno-microenvironment remodeling, play crucial roles in the cancer progression process and has become the hotspot of cancer research field in recent years. Nowadays, the exact molecular mechanisms of cancer stemness have not been fully understood. Extensive studies have recently implicated that non-coding RNA (ncRNA) plays vital roles in modulating cancer stemness. Notably, N6-methyladenosine (m6A) modification is of crucial importance for RNAs to exert their biological functions, including RNA splicing, stability, translation, degradation, and export. Emerging evidence has revealed that m6A modification can govern the expressions and functions of ncRNAs, consequently controlling cancer stemness properties. However, the interaction mechanisms between ncRNAs and m6A modification in cancer stemness modulation are rarely investigated. In this review, we elucidate the recent findings on the relationships of m6A modification, ncRNAs, and cancer stemness. We also focus on some key signaling pathways such as Wnt/β-catenin signaling, MAPK signaling, Hippo signaling, and JAK/STAT3 signaling to illustrate the underlying interplay mechanisms between m6A modification and ncRNAs in cancer stemness. In particular, we briefly highlight the clinical potential of ncRNAs and m6A modifiers as promising biomarkers and therapeutic targets for indicating cancer stemness properties and improving the diagnostic precision for a wide variety of cancers.
    Keywords:  N6-methyladenosine (m6A) modification; biomarkers; cancer stemness; non-coding RNA (ncRNA); signaling pathways
  9. Cells. 2021 Jul 02. pii: 1669. [Epub ahead of print]10(7):
      KRAS and TP53 mutations are the two most common driver mutations in patients with lung adenocarcinoma (LUAD), and they appear to reduce latency and increase metastatic proclivity when a KRAS and TP53 co-mutation (KRAS/TP53-mut) occurs. However, the molecular mechanism involved is unclear. N6-methyladenosine (m6A), the most abundant RNA modification in mammal mRNAs, plays a critical role in tumorigenesis. Here, we used genomic and transcriptomic data and found that only LUAD patients with KRAS/TP53-mut, but not an individual mutation, appeared to exhibit poor overall survival when compared with patients without KRAS and TP53 mutation (wildtype). Subsequently, we analyzed the differential expression of the 15-m6A-related genes in LUAD with different mutations and found that YTHDF1 was the most upregulated in KRAS/TP53-mut patients and associated with their adverse prognosis. Bioinformatics and experimental evidence indicated that elevated YTHDF1 functionally promoted the translation of cyclin B1 mRNA in an m6A-dependent manner, thereby facilitating the tumor proliferation and poor prognosis of LUAD with KRAS/TP53-mut. Furthermore, the concurrent increase in YTHDF1 and cyclin B1 was confirmed by immunohistochemistry staining in patients with co-occurring KRAS/TP53 mutations. YTHDF1 was correlated with an unfavorable clinical stage and tumor size. Collectively, we identified and confirmed a novel "YTHDF1-m6A-cyclin B1 translation" axis as an essential molecular pathway for the prognosis of KRAS/TP53-mut LUAD.
    Keywords:  KRAS/TP53 mutation; YTHDF1; cyclin B1; lung adenocarcinoma; m6A modification
  10. Front Pharmacol. 2021 ;12 667644
      Background: The total flavones of Abelmoschus manihot (TFA), a compound that is extracted from Abelmoschus manihot, has been widely used in China to reduce podocyte injury in diabetic kidney disease (DKD). However, the mechanisms underlying the therapeutic action of this compound have yet to be elucidated. Podocyte pyroptosis is characterized by activation of the NLRP3 inflammasome and plays an important role in inflammation-mediated diabetic kidneys. Regulation of the PTEN/PI3K/Akt pathway is an effective strategy for improving podocyte damage in DKD. Previous research has also shown that N6-methyladenosine (m6A) modification is involved in DKD and that m6A-modified PTEN regulates the PI3K/Akt pathway. In this study, we investigated whether TFA alleviates podocyte pyroptosis and injury by targeting m6A modification-mediated NLRP3-inflammasome activation and PTEN/PI3K/Akt signaling. Methods: We used MPC-5 cells under high glucose (HG) conditions to investigate the key molecules that are involved in podocyte pyroptosis and injury, including activation of the NLRP3 inflammasome and the PTEN/PI3K/Akt pathway. We detected alterations in the levels of three methyltransferases that are involved in m6A modification. We also investigated changes in the levels of these key molecules in podocytes with the overexpression or knockdown of methyltransferase-like (METTL)3. Results: Analysis showed that TFA and MCC950 protected podocytes against HG-induced pyroptosis and injury by reducing the protein expression levels of gasdermin D, interleukin-1β, and interleukin-18, and by increasing the protein expression levels of nephrin, ZO-1, WT1 and podocalyxin. TFA and 740Y-P inhibited activation of the NLRP3 inflammasome via the PI3K/Akt pathway by inhibiting the protein levels of NIMA-related kinase7, NLRP3, ASC, and caspase-1, and by increasing the protein expression levels of p-PI3K and p-Akt. TFA improved pyroptosis and injury in HG-stimulated podocytes by regulating METTL3-dependent m6A modification. Conclusion: Collectively, our data indicated that TFA could ameliorate pyroptosis and injury in podocytes under HG conditions by adjusting METTL3-dependent m6A modification and regulating NLRP3-inflammasome activation and PTEN/PI3K/Akt signaling. This study provides a better understanding of how TFA can protect podocytes in DKD.
    Keywords:  NLRP3-inflammasome activation; PTEN/PI3K/Akt signaling; diabetic kidney disease; m6A modification; podocyte pyroptosis; total flavones of Abelmoschus manihot
  11. Exp Cell Res. 2021 Jul 31. pii: S0014-4827(21)00314-1. [Epub ahead of print]406(1): 112761
      Stresses, such as neurohumoral activation, induced pathological cardiac hypertrophy is the main risk factor for heart failure. The ubiquitin-proteasome system (UPS) plays a key role in maintaining protein homeostasis and cardiac function. However, research on the role and mechanism of deubiquitinating enzymes (DUBs) in cardiac hypertrophy is limited. Here, we observe that the deubiquitinating enzyme ubiquitin-specific protease 12(USP12) is upregulated in Ang II-induced hypertrophic hearts and primary neonatal rat cardiomyocytes (NRCMs). Inhibition of USP12 ameliorate Ang II-induced myocardial hypertrophy, while overexpression of USP12 have the opposite effect. USP12 deficiency also significantly attenuate the phenotype of Ang II-induced cardiac hypertrophy in vivo. Moreover, we demonstrate that USP12 aggravate Ang II-induced cardiac hypertrophy by enhancing METTL3, a methyltransferase which catalyze N6-methyladenosine (m6A) modification on messenger RNA and acts as a harmful factor in pathological cardiac hypertrophy. Upregulation of METTL3 reverse the reduction of myocardial hypertrophy induced by USP12 silencing in NRCMs. In contrast, knockdown of METTL3 attenuate the aggravation of myocardial hypertrophy in USP12-overexpressing NRCMs. Furthermore, we discover that USP12 promote the expression of METTL3 via upregulating p300. Mechanistically, USP12 binds and stabilizes p300, thereby activating the transcription of its downstream gene METTL3. Finally, our data show that USP12 is partially dependent on the stabilization of p300 to activate METTL3 expression and promote myocardial hypertrophy. Taken together, our results demonstrate that USP12 acts as a pro-hypertrophic deubiquitinating enzyme via enhancing p300/METTL3 axis, indicating that targeting USP12 could be a potential treatment strategy for pathological cardiac hypertrophy.
    Keywords:  Deubiquitinating enzyme; METTL3; Pathological cardiac hypertrophy; USP12; p300
  12. Pathol Res Pract. 2021 Jul 13. pii: S0344-0338(21)00211-9. [Epub ahead of print]225 153550
      BACKGROUND: N6-methyladenosine (m6A), a common internal modification on RNAs, has been found to be closely linked with RNA biosynthesis/metabolism and cancer development. In this text, the roles and molecular mechanisms of m6A-bind protein IGF2BP2 in the development of thyroid cancer (TC) were investigated in vitro.METHODS: IGF2BP2 and lncRNA HAGLR were screened out through multiple public databases such as TCGA, Ualcan, POSTAR2, Starbase, and GEPIA. Cell proliferative, migratory and invasive abilities were assessed by CCK-8, Transwell migration and invasion assays, respectively. Cell cycle distribution and cell apoptotic patterns were measured by flow cytometry. The interaction between HAGLR and IGF2BP2 was examined by RIP, RNA pull-down and luciferase assays and bioinformatics analysis. The effect of IGF2BP2 knockdown on the m6A level of HAGLR was explored by meRIP assay.
    RESULTS: IGF2BP2 was highly expressed in TC tumor tissues. IGF2BP2 knockdown weakened cell proliferative, migratory, and invasive abilities, and induced cell cycle arrest and cell apoptosis in TC cells. LncRNA HAGLR expression was markedly upregulated and positively associated with IGF2BP2 expression in TC tissues. IGF2BP2 knockdown reduced HAGLR expression and transcript stability in TC cells. IGF2BP2 regulated HAGLR expression in an m6A-dependent manner. HAGLR overexpression weakened the effects of IGF2BP2 loss on cell proliferation, migration, invasion, apoptosis, and cell cycle progression in TC cells.
    CONCLUSION: IGF2BP2 loss inhibited cell proliferation, migration and invasion, and induced cell apoptosis and cell cycle arrest by down-regulating HAGLR expression in an m6A-dependent manner in TC cells, providing some potential diagnostic and therapeutic targets for TC.
    Keywords:  HAGLR; IGF2BP2; LncRNA; N6-methyladenosine; Thyroid cancer
  13. Cancer Res. 2021 Aug 06. pii: canres.0494.2021. [Epub ahead of print]
      Pseudogenes may play important roles in cancer. Here, we explore the mechanism and function of a pseudogene WTAPP1 in the progress of pancreatic ductal adenocarcinoma (PDAC). WTAPP1 RNA was significantly elevated in PDAC and was associated with poor prognosis in patients. Overexpression of WTAPP1 RNA promoted PDAC proliferation and invasiveness in vitro and in vivo. Mechanistically, N6-methyladenosine (m6A) modification stabilized WTAPP1 RNA via CCHC-type zinc finger nucleic acid binding protein (CNBP), resulting in increased levels of WTAPP1 RNA in PDAC cells. Excessive WTAPP1 RNA bound its protein-coding counterpart WT1 associated protein (WTAP) mRNA and recruited more EIF3 translation initiation complex to promote WTAP translation. Increased WTAP protein enhanced the activation of Wnt signaling and provoked the malignant phenotypes of PDAC. Decreasing WTAPP1 RNA significantly suppressed the in vivo growth and metastasis of PDAC cell lines and patient-derived xenografts. These results indicate that m6A-mediated increases in WTAPP1 expression promotes PDAC progression and thus may serve as a therapeutic target.
  14. Front Cell Dev Biol. 2021 ;9 703629
      Background: Colon adenocarcinoma (COAD) is the most common type of colon cancer. To date, however, the prognostic values of m6A RNA methylation-related long non-coding RNAs (lncRNAs) in COAD are largely unknown.Materials and Methods: The m6A-related lncRNAs were identified from The Cancer Genome Atlas (TCGA) data set. Univariate and multivariate Cox regression analyses were performed to explore the prognostic m6A-related lncRNAs. Consistent clustering analysis was performed to classify the COAD patients into different subgroups based on the expression of m6A-related lncRNAs. The potential biological functions as well as differences in the stemness index and tumor immune microenvironment between different subgroups were analyzed. The prognostic m6A-related lncRNAs were used to establish an m6A-related lncRNA risk model to predict prognosis and survival status.
    Results: We identified 31 m6A-associated lncRNAs with prognostic values from the TCGA data set. Based on the expression of prognostic m6A-associated lncRNAs, TCGA-COAD patients were classified into three clusters using consistent clustering analysis. There was a low correlation of tumor stemness between the three clusters but a significant correlation with the tumor immune microenvironment as well as the tumor mutational load. Thirty-one prognostic-related m6A-associated lncRNAs were used to construct a risk model, which was further determined by survival analysis, receiver operating characteristic (ROC) curve, and univariate and multifactor Cox analysis. The m6A-related risk model demonstrates good performance in predicting prognosis and survival status. The model-based high-risk group exhibited poorer overall survival (OS) compared with the low-risk group.
    Conclusion: In this study, we construct a risk model that consists of 31 m6A-related lncRNAs with independent prognostic values in COAD. Our study shows the critical roles of these 31 m6A-related lncRNAs in the tumor immune microenvironment, indicating the prospect of informing prognostic stratification and the development of immunotherapeutic strategies for COAD patients.
    Keywords:  M6A; colon adenocarcinoma; lncRNA; prognosis; tumor immune microenvironment
  15. Cancer Biol Med. 2021 Aug 04. pii: j.issn.2095-3941.2020.0661. [Epub ahead of print]
      OBJECTIVE: Investigation of the regulatory mechanisms of cell stemness in cholangiocarcinoma (CCA) is essential for developing effective therapies to improve patient outcomes. The purpose of this study was to investigate the function and regulatory mechanism of m6A modifications in CCA cell stemness.METHODS: Interleukin 6 (IL-6) treatment was used to induce an inflammatory response, and loss-of-function studies were conducted using mammosphere culture assays. Chromatin immunoprecipitation, polysome profiling, and methylated RNA immunoprecipitation analyses were used to identify signaling pathways. The in vitro findings were verified in a mice model.
    RESULTS: We first identified that m6A writers were highly expressed in CCAs and further showed that STAT3 directly bound to the gene loci of m6A writers, showing that IL-6/STAT3 signaling regulated expressions of m6A writers. Downregulating m6A writers prevented cell proliferation and migration in vitro and suppressed CCA tumorigenesis in vivo. Notably, the knockdown of m6A writers inhibited CCA cell stemness that was triggered by IL-6 treatment. Mechanistically, IGF2BP2 was bound to CTNNB1 transcripts, significantly enhancing their stability and translation, and conferring stem-like properties. Finally, we confirmed that the combination of m6A writers, IGF2BP2, and CTNNB1 distinguished CCA tissues from normal tissues.
    CONCLUSIONS: Overall, this study showed that the IL-6-triggered inflammatory response facilitated the expressions of m6A writers and cell stemness in an m6A-IGF2BP2-dependent manner. Furthermore, the study showed that m6A modification was a targetable mediator of the response to inflammation factor exposure, was a potential diagnostic biomarker for CCA, and was critical to the progression of CCA.
    Keywords:  Cholangiocarcinoma; IGF2BP2; IL-6; N6-methyladenosine (m6A); cell stemness
  16. Front Oncol. 2021 ;11 679634
      Background: Chronic myeloid leukemia (CML) is an acquired hematopoietic stem malignant disease originating from the myeloid system. Long non-coding RNAs (lncRNAs) have been widely explored in cancer tumorigenesis. However, their roles in CML remain largely unclear.Methods: The peripheral blood mononuclear cells (PBMCs) and CML cell lines (K562, KCL22, MEG01, BV173) were collected for in vitro research. Real-time quantitative polymerase chain reaction was used to determine the mRNA expression levels. Cell viability and apoptosis were analyzed by cell counting kit 8 and flow cytometry assays. The targeting relationships were predicted using Starbase and TargetScan and ulteriorly verified by RNA pull-down and luciferase reporter assays. Western blotting assay was performed to assess the protein expressions. N6-methyladenosine (m6A) modification sites were predicted by SRAMP and confirmed by Methylated RNA immunoprecipitation (MeRIP) assay.
    Results: LncRNA nuclear-enriched abundant transcript 1 (NEAT1) expression levels were decreased in the CML cell lines and PBMCs of CML patients. Moreover, METTL3-mediated m6A modification induced the aberrant expression of NEAT1 in CML. Overexpression of NEAT1 inhibited cell viability and promoted the apoptosis of CML cells. Additionally, miR-766-5p was upregulated in CML PBMCs and abrogated the effects of NEAT1 on cell viability and apoptosis of the CML cells. Further, CDKN1A was proved to be the target gene of miR-766-5p and was downregulated in the CML PBMCs. Knockdown of CDKN1A reversed the effects of NEAT1.
    Conclusion: The current research elucidates a novel METTL3/NEAT1/miR-766-5p/CDKN1A axis which plays a critical role in the progression of CML.
    Keywords:  CDKN1A; METTL3; N6-methyladenosine (m6A) modification; NEAT1; chronic myeloid leukemia; miR-766-5p
  17. Mol Cell. 2021 Jul 29. pii: S1097-2765(21)00542-6. [Epub ahead of print]
      The emerging "epitranscriptomics" field is providing insights into the biological and pathological roles of different RNA modifications. The RNA methyltransferase METTL1 catalyzes N7-methylguanosine (m7G) modification of tRNAs. Here we find METTL1 is frequently amplified and overexpressed in cancers and is associated with poor patient survival. METTL1 depletion causes decreased abundance of m7G-modified tRNAs and altered cell cycle and inhibits oncogenicity. Conversely, METTL1 overexpression induces oncogenic cell transformation and cancer. Mechanistically, we find increased abundance of m7G-modified tRNAs, in particular Arg-TCT-4-1, and increased translation of mRNAs, including cell cycle regulators that are enriched in the corresponding AGA codon. Accordingly, Arg-TCT expression is elevated in many tumor types and is associated with patient survival, and strikingly, overexpression of this individual tRNA induces oncogenic transformation. Thus, METTL1-mediated tRNA modification drives oncogenic transformation through a remodeling of the mRNA "translatome" to increase expression of growth-promoting proteins and represents a promising anti-cancer target.
    Keywords:  Arg-TCT; METTL1; N(7)-methylguanosine; cancer; m(7)G; oncogene; tRNA; translation
  18. J Oncol. 2021 ;2021 7488188
      Abnormal m6A methylation plays a significant role in cancer progression. Increasingly, researchers have focused on developing lncRNA signatures to evaluate the prognosis of cancer patients. The specific function of m6A-related lncRNAs in the prognosis of bladder cancer patients and the immune microenvironment of bladder cancer remains elusive. Herein, we performed a comprehensive analysis of m6A-related lncRNA prognostic values and their association with the immune microenvironment in bladder cancer using the TCGA dataset. A total of 9 m6A-related lncRNAs were dramatically correlated with overall survival outcomes in bladder cancer. Two molecular subtypes (cluster 1 and cluster 2) were identified by consensus clustering for 9 m6A-related prognostic lncRNAs. Cluster 1 was significantly correlated with poor prognosis, advanced clinical stage, higher PD-L1 expression, a higher ESTIMATEScore and immuneScore, and distinct immune cell infiltration. GSEA revealed the enrichment of apoptosis and the JAK-STAT signaling pathway in cluster 2. A prognostic risk score was constructed using 9 m6A-related prognostic lncRNAs, which functioned as an independent prognostic factor for bladder cancer. Moreover, bladder cancer patients in the low-risk score group had a higher pN stage, pT stage, and clinical stage and a lower tumor grade and immuneScore. The risk score was correlated with the infiltration levels of certain immune cells, including B cells, plasma cells, follicular helper T cells, regulatory T cells, resting NK cells, neutrophils, M0 macrophages, M1 macrophages, and M2 macrophages. Collectively, our study elucidated the important role of m6A-related lncRNAs in the prognosis of bladder cancer patients and in the bladder cancer immune microenvironment. The results suggest that the components of the m6A-related prognostic lncRNA signature might serve as a crucial mediator of the immune microenvironment in bladder cancer, representing promising therapeutic targets for improving immunotherapeutic efficacy.
  19. Biol Proced Online. 2021 Aug 01. 23(1): 15
      AIM: To illustrate the influence of N6-methyladenosine long non-coding RNAs and immune cell infiltration in gastric cancer.METHODS: We downloaded workflow-type data and clinical data from The Cancer Genome Atlas project. The relationship of lncRNA and m6A was identified. Kyoto Encyclopedia of Genes and Genomes gene expression enrichment analysis was performed. Lasso regression was utilized to construct a prognostic model. Survival analysis to explore the relationship between m6A lncRNA and clinical survival data. Differential analysis of the tumor microenvironment and immune correlation analysis to determine immune cell infiltration levels and their correlation with clinical prognosis.
    RESULTS: Co-expression analysis indicated that lncRNA expression was associated closely with m6A. m6A-lncRNAs were partially highly expressed in tumor tissue and could be used in a prognostic model to predict GC prognosis, independent of other clinical characteristics. "ADIPPOCYTOKINE SIGNALING PATHWAY" was most significantly enriched according to GSEA. ACBD3-AS1 was overexpressed in tumor tissue. Naïve B cell, Plasma cells, resting CD4 memory T cell were highly infiltrated tissues in cluster 2, while Macrophages M2, resting Mast cells, Monocytes, regulates T cells were lowly in cluster 1. All related scores were higher in cluster 2, indicating a lower purity of tumor cells and higher density of immune-related cells in the tumor microenvironment.
    CONCLUSION: m6A lncRNA is closely related to the occurrence and progression of GC. The corresponding prognostic model can be utilized to evaluate the prognosis of GC. m6A lncRNA and related immune cell infiltration in the tumor microenvironment can provide novel therapeutic targets for further research.
    Keywords:  Bioinformatics analysis; Gastric cancer; Immune cell infiltration; N6-methyladenosine; lncRNAs
  20. Sci Adv. 2021 Aug;pii: eabd2605. [Epub ahead of print]7(32):
      The study of RNA modifications in large clinical cohorts can reveal relationships between the epitranscriptome and human diseases, although this is especially challenging. We developed ModTect (, a statistical framework to identify RNA modifications de novo by standard RNA-sequencing with deletion and mis-incorporation signals. We show that ModTect can identify both known (N 1-methyladenosine) and previously unknown types of mRNA modifications (N 2,N 2-dimethylguanosine) at nucleotide-resolution. Applying ModTect to 11,371 patient samples and 934 cell lines across 33 cancer types, we show that the epitranscriptome was dysregulated in patients across multiple cancer types and was additionally associated with cancer progression and survival outcomes. Some types of RNA modification were also more disrupted than others in patients with cancer. Moreover, RNA modifications contribute to multiple types of RNA-DNA sequence differences, which unexpectedly escape detection by Sanger sequencing. ModTect can thus be used to discover associations between RNA modifications and clinical outcomes in patient cohorts.
  21. Oncogene. 2021 Aug 03.
      5-Methylcytosine (m5C) is a posttranscriptional RNA modification participating in many critical bioprocesses, but its functions in human cancer remain unclear. Here, by detecting the transcriptome-wide m5C profiling in esophageal squamous cell carcinoma (ESCC), we showed increased m5C methylation in ESCC tumors due to the overexpressed m5C methyltransferase NSUN2. Aberrant expression of NSUN2 was positively regulated by E2F Transcription Factor 1 (E2F1). High NSUN2 levels predicted poor survival of ESCC patients. Moreover, silencing NSUN2 suppressed ESCC tumorigenesis and progression in Nsun2 knockout mouse models. Mechanistically, NSUN2 induced m5C modification of growth factor receptor-bound protein 2 (GRB2) and stabilized its mRNA, which was mediated by a novel m5C mediator, protein lin-28 homolog B (LIN28B). Elevated GRB2 levels increased the activation of PI3K/AKT and ERK/MAPK signalling. These results demonstrate that NSUN2 enhances the initiation and progression of ESCC via m5C-LIN28B dependent stabilization of GRB2 transcript, providing a promising epitranscriptomic-targeted therapeutic strategy for ESCC.
  22. Steroids. 2021 Aug 03. pii: S0039-128X(21)00109-4. [Epub ahead of print] 108897
      Obesity and visceral adiposity are major risk factors for type 2 diabetes mellitus (T2DM). The fat mass and obesity-associated (FTO) gene is associated with increased risk of obesity and T2DM. The aim of this work was to study the association between FTO gene expression and serum FTO protein level with disease severity in T2DM patients.PATIENTS AND METHODS: One hundred T2DM patients were divided into two equal groups according to diabetes control and complications and fifty healthy controls were included in this study. FTO messenger ribonucleic acid (mRNA) expression level was analyzed by Real time polymerase chain reaction (PCR) technique and serum level of FTO protein was measured by ELISA.
    RESULTS: FTO gene expression and FTO protein levels were increased in the two T2DM groups compared to the control group with significant further increases in patients with severe disease. FTO gene expression and FTO protein levels were positively correlated with obesity, insulin resistance and blood glucose indices as well as the presence of diabetic complications. Regression analyses showed that FTO gene expression and FTO protein levels were risk factors for T2DM severity.
    CONCLUSIONS: Increased FTO gene expression and its serum protein levels are associated with increased T2DM severity.
    Keywords:  Fat-mass and obesity-associated gene; Insulin resistance; Obesity; Type2diabetes mellitus
  23. Mol Cell. 2021 Jul 29. pii: S1097-2765(21)00555-4. [Epub ahead of print]
      Cancer cells selectively promote translation of specific oncogenic transcripts to facilitate cancer survival and progression, but the underlying mechanisms are poorly understood. Here, we find that N7-methylguanosine (m7G) tRNA modification and its methyltransferase complex components, METTL1 and WDR4, are significantly upregulated in intrahepatic cholangiocarcinoma (ICC) and associated with poor prognosis. We further reveal the critical role of METTL1/WDR4 in promoting ICC cell survival and progression using loss- and gain-of-function assays in vitro and in vivo. Mechanistically, m7G tRNA modification selectively regulates the translation of oncogenic transcripts, including cell-cycle and epidermal growth factor receptor (EGFR) pathway genes, in m7G-tRNA-decoded codon-frequency-dependent mechanisms. Moreover, using overexpression and knockout mouse models, we demonstrate the crucial oncogenic function of Mettl1-mediated m7G tRNA modification in promoting ICC tumorigenesis and progression in vivo. Our study uncovers the important physiological function and mechanism of METTL1-mediated m7G tRNA modification in the regulation of oncogenic mRNA translation and cancer progression.
  24. Life (Basel). 2021 Jul 08. pii: 669. [Epub ahead of print]11(7):
      METTL16 methyltransferase is responsible for the methylation of N6-adenosine (m6A) in several RNAs. In mouse cells, we showed that the nuclear distribution of METTL16 is cell cycle-specific. In the G1/S phases, METTL16 accumulates to the nucleolus, while in the G2 phase, the level of METTL16 increases in the nucleoplasm. In metaphase and anaphase, there is a very low pool of the METTL16 protein, but in telophase, residual METTL16 appears to be associated with the newly formed nuclear lamina. In A-type lamin-depleted cells, we observed a reduction of METTL16 when compared with the wild-type counterpart. However, METTL16 does not interact with A-type and B-type lamins, but interacts with Lamin B Receptor (LBR) and Lap2α. Additionally, Lap2α depletion caused METTL16 downregulation in the nuclear pool. Furthermore, METTL16 interacted with DDB2, a key protein of the nucleotide excision repair (NER), and also with nucleolar proteins, including TCOF, NOLC1, and UBF1/2, but not fibrillarin. From this view, the METTL16 protein may also regulate the transcription of ribosomal genes because we observed that the high level of m6A in 18S rRNA appeared in cells with upregulated METTL16.
    Keywords:  METTL16; cell cycle; epitranscriptome; nucleolus; rDNA