bims-rimeca 2023-04-30 papers

bims-rimeca

Biomed News

on RNA methylation in cancer

Issue of 2023–04–30
twenty-one papers selected by
Sk Ramiz Islam, Saha Institute of Nuclear Physics

m6A Methyltransferase, WTAP, Promotes Cancer Progression in Laryngeal Squamous Cell Carcinoma by Regulating PLAU Stability.
N6-methyladenosine promotes translation of VEGFA to accelerate angiogenesis in lung cancer.
HIF1α-mediated transactivation of WTAP promotes AML cell proliferation via m6A-dependent stabilization of KDM4B mRNA.
METTL3 promotes the malignancy of non-small cell lung cancer by N6-methyladenosine modifying SFRP2.
METTL3 reduces oxidative stress-induced apoptosis in presbycusis by regulating the N6-methyladenosine level of SIRT1 mRNA.
N6-methyladenosine modification contributes to respiratory syncytial virus infection.
Post-transcriptional modification of m6A methylase METTL3 regulates ERK-induced androgen-deprived treatment resistance prostate cancer.
O-GlcNAcylation promotes the cytosolic localization of the m6A reader YTHDF1 and colorectal cancer tumorigenesis.
Upregulation of UBR1 m6A methylation by METTL14 inhibits autophagy in spinal cord injury.
YTHDF1 Promotes Bladder Cancer Cell Proliferation via the METTL3/YTHDF1-RPN2-PI3K/AKT/mTOR Axis.
Characterization of the m6A regulator-mediated methylation modification patterns in oral squamous cell carcinoma.
Alteration of m6A-Tagged RNA Profiles in Bone Originated from Periprosthetic Joint Infection.
m6A epitranscriptomic regulation of tissue homeostasis during primate aging.
METTL3 affects FLT3-ITD+ acute myeloid leukemia by mediating autophagy by regulating PSMA3-AS1 stability.
m6A-modification regulated circ-CCT3 acts as the sponge of miR-378a-3p to promote hepatocellular carcinoma progression.
Purine anabolism creates therapeutic vulnerability in hepatocellular carcinoma via m6A-mediated epitranscriptomic regulation.
IGF2BP2 promotes colorectal cancer progression by upregulating the expression of TFRC and enhancing iron metabolism.
[Knockdown of IGF2BP2 inhibits colorectal cancer cell proliferation, migration and promotes tumor immunity by down-regulating MYC expression].
5-Azacytidine promotes HCC cell metastasis by up-regulating RDH16 expression.
The activation of mTOR signalling modulates DNA methylation by enhancing DNMT1 translation in hepatocellular carcinoma.
MKRN3 inhibits puberty onset via interaction with IGF2BP1 and regulation of hypothalamic plasticity.

Ann Clin Lab Sci. 2023 Mar;53(2): 293-302

m6A Methyltransferase, WTAP, Promotes Cancer Progression in Laryngeal Squamous Cell Carcinoma by Regulating PLAU Stability.

Fangfang Liu, Fan Xia, Cancan Wang, Rongling Ren, Min Zhang, Jie Gong, Qiuying Zhang.

OBJECTIVE: Laryngeal squamous cell carcinoma (LSCC) is a malignancy originating from laryngeal squamous cell lesions. Wilm's tumor 1-associated protein (WTAP)-mediated N6-methyladenosine (m6A) modification has been verified to stimulate the progression of numerous cancers, except for LSCC. This study was aimed at exploring the role of WTAP and its mechanism of action in LSCC.
METHODS: The expression of WTAP and plasminogen activator urokinase (PLAU) mRNAs in LSCC tissues and cells was quantified using qRT-PCR. Western blotting was performed to estimate PLAU levels in LSCC cells. The relationship between WTAP and PLAU was ascertained using luciferase reporter and methylated-RNA immunoprecipitation (Me-RIP) assays. Functionally, the interaction of WTAP with PLAU in LSCC cells was investigated using CCK-8, EdU, and Transwell assays.
RESULTS: The expression of WTAP and PLAU was increased in LSCC, and was positively correlated. WTAP regulated PLAU stability in an m6A-dependent manner. WTAP deficiency suppressed the migration, invasion, and proliferation of LSCC cells. Overexpression of PLAU rescued the phenotype induced by WTAP knockdown in vitro.
CONCLUSIONS: These results indicate that WTAP mediates the m6A modification of PLAU to accelerate the growth, migration, and invasion of cells in LSCC. To our knowledge, this is the first report to clarify the functions of WTAP in LSCC and the underlying mechanisms in detail. Based on these findings, we suggest that WTAP may serve as a therapeutic target for LSCC.

Keywords: Laryngeal squamous cell carcinoma; N6-methyladenosine; PLAU; invasion; migration; proliferation
Cancer Res. 2023 Apr 27. pii: CAN-22-2449. [Epub ahead of print]

N6-methyladenosine promotes translation of VEGFA to accelerate angiogenesis in lung cancer.

Haisheng Zhang, Jiawang Zhou, Jiexin Li, Zhaotong Wang, Zhuojia Chen, Ziyan Lv, Lichen Ge, Guoyou Xie, Guoming Deng, Yalan Rui, Hongbing Huang, Likun Chen, Hongsheng Wang.

Angiogenesis is hijacked by cancer to support tumor growth. RNA modifications such as N6-methyladenosine (m6A) can regulate several aspects of cancer, including angiogenesis. Here, we find that m6A triggers angiogenesis in lung cancer by upregulating vascular endothelial growth factor-A (VEGFA), a central regulator of neovasculature and blood vessel growth. m6A-sequencing and functional studies confirmed that m6A modification of the 5'UTR of VEGFA positively regulates its translation. Specifically, methylation of a 5'UTR internal ribosome entry site (IRES) recruited the YTHDC2/eIF4GI complex to trigger cap-independent translation initiation. Intriguingly, the m6A methylation site A856 of the 5'UTR was located within the conserved upstream open reading frame (uORF) of VEGFA IRES-A, which overcomes uORF-mediated translation suppression while facilitating G-quadruplex-induced translation of VEGFA. Targeted specific demethylation of VEGFA m6A significantly decreased expression of VEGFA and reduced lung cancer cell-driven angiogenesis. In vivo and clinical data confirmed the positive effects of m6A modification of VEGFA on angiogenesis and tumor growth of lung cancer. This study not only reveals that the m6A/VEGFA axis is a potential target for lung cancer therapy but also expands our understanding of the impact of m6A modification of IRES in the 5'UTR of mRNA on translation regulation.

DOI: https://doi.org/10.1158/0008-5472.CAN-22-2449
Leukemia. 2023 Apr 22.

HIF1α-mediated transactivation of WTAP promotes AML cell proliferation via m6A-dependent stabilization of KDM4B mRNA.

Yang-Liu Shao, Yu-Qing Li, Meng-Yue Li, Li-Li Wang, Hui-Sheng Zhou, Dai-Hong Liu, Li Yu, Ji Lin, Xiao-Ning Gao.

Hypoxia inducible factor 1α (HIF1α) is abnormally overexpressed in t(8;21) acute myeloid leukemia (AML) and functions as an oncogene through transactivating DNA methyltransferase 3 alpha leading to DNA hypermethylation. However, it remains unclear whether HIF1α influences RNA N6-methyladenosine (m6A) methyltransferases. Here, we show that HIF1α promotes the expression of Wilms tumor 1-associated protein (WTAP), a main component of the m6A methyltransferase complex, markedly alters the transcriptome-wide m6A distribution and enhances cell proliferation in t(8;21) AML. In agreement with this, WTAP is overexpressed and predicts poor prognosis in t(8;21) AML patients. Moreover, WTAP knockdown inhibits growth, and induces apoptosis and differentiation of leukemia cells. Mechanistically, HIF1α transactivates WTAP gene expression by directly binding to the hypoxia-response element of its promoter region. Pharmacological or genetic intervention in the HIF1α-WTAP axis results in the reduction of m6A level on lysine demethylase 4B (KDM4B) transcripts and increased its degradation, correlated with lower expression of KDM4B and higher trimethylation levels of histone H3 on lysine 9. KDM4B knockdown inhibits leukemia cell growth in vitro and in mice. Thus, HIF1α-mediated WTAP high expression enhances the malignant behavior of leukemia cells and drives a crosstalk between m6A RNA methylation and histone methylation through monitoring m6A-dependant KDM4B translation.

DOI: https://doi.org/10.1038/s41375-023-01904-1
Cancer Gene Ther. 2023 Apr 27.

METTL3 promotes the malignancy of non-small cell lung cancer by N6-methyladenosine modifying SFRP2.

Shu Zhao, Peng Song, Gang Zhou, Dong Zhang, Yi Hu.

This study aimed to investigate the roles of METTL3, a regulator of m6A, in NSCLC. RT-qPCR was applied to determine mRNA of m6A-associated genes and SFRP2, and western blot were used for ZEB1 and MMP9 protein expression. Total m6A level was measured using methylated RNA immunoprecipitation (MeRIP) assay, and RIP was used to access m6A level of SFRP2. Cellular behaviors were detected using CCK-8 and tranwell assays. Xenograft assays were conducted to further verify the roles of METTL3 and SFRP2 in NSCLC. The expression level of METTL3 was higher in NSCLC than normal controls. However, downregulation of METTL3 restrained the proliferation, migration and invasion of NSCLC cells. Enhanced expression of METTL3 caused the inverse consequences. Moreover, SFRP2 was found to be negatively regulated by METTL3. Intriguingly, the anti-tumor functions of METTL3 knockdown in the phenotype of NSCLC cells and xenograft mice were overturned by inhibition of SFRP2. Silencing METTL3 resulted in the enhanced stability of SFRP2. Finally, downregulation of SFRP2 induced by METTL3 activated the Wnt/β-catenin signaling pathway in NSCLC. METTL3 acted as an oncogene in the pathogenesis of NSCLC via suppressing SFRP2 to activate Wnt/β-catenin signaling pathway, indicating that METTL3 might be a promising predictor in NSCLC.

DOI: https://doi.org/10.1038/s41417-023-00614-1
Neuroscience. 2023 Apr 20. pii: S0306-4522(23)00117-3. [Epub ahead of print]

METTL3 reduces oxidative stress-induced apoptosis in presbycusis by regulating the N6-methyladenosine level of SIRT1 mRNA.

Enhao Wang, Yang Li, Hejie Li, Yingzhao Liu, Ruijie Ming, Jiahui Wei, Peiyu Du, Xiangrui Li, Shimin Zong, Hongjun Xiao.

  N6-methyl adenosine (m6A) modification is known to play a crucial role in various aging-related diseases. However, its involvement in presbycusis, a type of age-related hearing loss, is not yet clear. We examined the changes in oxidative stress levels in both plasma of presbycusis patients and mice. To determine the expression of m6A and its functional enzymes, we used liquid chromatography tandem-mass spectrometry (LC-MS/MS), enzyme-linked immunosorbent assay (ELISA), and RT-PCR to analyze the total RNA of presbycusis patients blood cells (n=8). Additionally, we detected the expression of m6A functional enzymes in the cochlea of presbycusis mice using immunohistochemistry. We assessed the effects of m6A methyltransferase METTL3 on SIRT1 protein expression, reactive oxygen species (ROS) levels, and apoptosis in an oxidative stress model of organ of Corti 1 (OC1) cells. To observe the effect on SIRT1 protein expression, we interfered with the m6A recognition protein IGF2BP3 using siRNA. In both presbycusis patients and mice, there was an increased level of oxidative stress in plasma.There was a decrease in the expression of m6A, METTL3, and IGF2BP3 in presbycusis patients blood cells. The expression of METTL3 and IGF2BP3 was also reduced in the cochlea of presbycusis mice. In OC1 cells, METTL3 positively regulated SIRT1 protein levels, while reversely regulated the level of ROS and apoptosis. IGF2BP3 was found to be involved in the regulation of SIRT1 protein expression. Moreover, METTL3 might played a protective role in oxidative stress-induced injury of OC1 cells, while both METTL3 and IGF2BP3 cooperatively regulated the m6A level and fate of SIRT1 mRNA in OC1 cells.

Keywords:  IGF2BP3; METTL3; Oxidative stress; Presbycusis; SIRT1; m6A

DOI:  https://doi.org/10.1016/j.neuroscience.2023.03.001
Heliyon. 2023 Apr;9(4): e15307

N6-methyladenosine modification contributes to respiratory syncytial virus infection.

Zhu Li, Yi Liu, Ling Zhang, Jiahua Tian, Hongping Wang, Hongwei Ding, Jin Nie, Hang Pi, Bingyao Wang, Daishun Liu.

   Background: Respiratory syncytial virus (RSV) is the second leading cause of death due to lower respiratory tract infections. Effective prevention and treatment measures are lacking, posing a huge socioeconomic burden to the world. N 6-methyladenosine (m6A) is the most common internal modification in messenger RNA and noncoding RNA. Numerous recent studies have shown that the dysregulation of m6A modification is associated with diseases caused by pathogenic viruses.
Methods: The changes in m6A modification were evaluated using m6A RNA methylation assay. The differences in gene expression levels of various m6A-modifying enzymes were observed using Quantitative Real-time PCR (qRT-PCR) during RSV infection. The autophagosomes were observed using transmission electron microscopy, and the expression of autophagy-associated protein Microtubule Associated Protein 1 Light Chain 3 Beta Ⅱ/Ⅰ (LC3B Ⅱ/Ⅰ) and Beclin1 in Human Normal Lung Epithelial Cells (BEAS-2B) cells using Western blot during RSV infection. The significantly differentially expressed genes were screened guided by bioinformatics. Their relationship with m6A-modifying enzymes was analyzed through protein-protein interaction network and expression correlation analysis.
Results: The m6A abundance decreased and demethylase Fat Mass and Obesity- Associated Protein (FTO) significantly increased during RSV infection after 24 h. We also found that the DNA Damage-Inducible Transcript 3 Protein (DDIT3) level significantly increased during RSV infection after 24 h and observed autophagosomes in BEAS-2B cells. In addition, RSV infection could cause the upregulation of LC3B Ⅱ/Ⅰ and Beclin1. The expression correlation analysis showed that DDIT3 levels were positively correlated with the FTO level, and Methyltransferase Like 3 (METTL3), RNA Binding Motif Protein 15B (RBM15B), YTH Domain-Containing Family Protein 1 (YTHDF1), and levels were negatively correlated with the DDIT3 level.
Conclusions: We uncovered a significant role for m6A modification during RSV infection. Also, a correlation was found between m6A and autophagy, providing new ideas for therapeutic advancements in RSV treatment.

Keywords:  Autophagy; Bioinformatics; Interaction; N6-methyladenosine; RSV

DOI:  https://doi.org/10.1016/j.heliyon.2023.e15307
Cell Death Dis. 2023 Apr 24. 14(4): 289

Post-transcriptional modification of m6A methylase METTL3 regulates ERK-induced androgen-deprived treatment resistance prostate cancer.

Yang Li, Shimiao Zhu, Yutong Chen, Qianwang Ma, Duo Kan, Wenyue Yu, Boya Zhang, Xuanrong Chen, Wanqing Wei, Yi Shao, Keruo Wang, Mingpeng Zhang, Shu Deng, Yuanjie Niu, Zhiqun Shang.

As the most common modification of RNA, N6-methyladenosin (m6A) has been confirmed to be involved in the occurrence and development of various cancers. However, the relationship between m6A and castration resistance prostate cancer (CRPC), has not been fully studied. By m6A-sequencing of patient cancer tissues, we identified that the overall level of m6A in CRPC was up-regulated than castration sensitive prostate cancer (CSPC). Based on the analysis of m6A-sequencing data, we found m6A modification level of HRas proto-oncogene, GTPase (HRAS) and mitogen-activated protein kinase kinase 2 (MEK2 or MAP2K2) were enhanced in CRPC. Specifically, tissue microarray analysis and molecular biology experiments confirmed that METTL3, an m6A "writer" up-regulated after castration, activated the ERK pathway to contribute to malignant phenotype including ADT resistance, cell proliferation and invasion. We revealed that METTL3-mediated ERK phosphorylation by stabilizing the transcription of HRAS and positively regulating the translation of MEK2. In the Enzalutamide-resistant (Enz-R) C4-2 and LNCap cell line (C4-2R, LNCapR) established in the current study, the ERK pathway was confirmed to be regulated by METTL3. We also found that applying antisense oligonucleotides (ASOs) to target the METTL3/ERK axis can restore Enzalutamide resistance in vitro and in vivo. In conclusion, METTL3 activated the ERK pathway and induced the resistance to Enzalutamide by regulating the m6A level of critical gene transcription in the ERK pathway.

DOI: https://doi.org/10.1038/s41419-023-05773-5
J Biol Chem. 2023 Apr 20. pii: S0021-9258(23)01766-0. [Epub ahead of print] 104738

O-GlcNAcylation promotes the cytosolic localization of the m6A reader YTHDF1 and colorectal cancer tumorigenesis.

Jie Li, Muhammad Ahmad, Lei Sang, Yahui Zhan, Yibo Wang, Yonghong Yan, Yue Liu, Weixiao Mi, Mei Lu, Yu Dai, Rou Zhang, Meng-Qiu Dong, Yun-Gui Yang, Xiaohui Wang, Jianwei Sun, Jing Li.

  O-linked N-acetylglucosamine (O-GlcNAc) is an emerging post-translation modification that couples metabolism with cellular signal transduction by crosstalk with phosphorylation and ubiquitination to orchestrate various biological processes. The mechanisms underlying the involvement of O-GlcNAc modifications in N6-methyladenosine (m6A) regulation are not fully characterized. Herein we show that O-GlcNAc modifies the m6A mRNA reader YTHDF1 and fine-tunes its nuclear translocation by the exportin protein Crm1. First we present evidence that YTHDF1 interacts with the sole O-GlcNAc transferase (OGT). Second, we verified Ser196/Ser197/Ser198 as the YTHDF1 O-GlcNAcylation sites, as described in numerous chemoproteomic studies. Then we constructed the O-GlcNAc-deficient YTHDF1-S196A/S197F/S198A (AFA) mutant, which significantly attenuated O-GlcNAc signals. Moreover, we revealed that YTHDF1 is a nucleocytoplasmic protein, whose nuclear export is mediated by Crm1. Furthermore, O-GlcNAcylation increases the cytosolic portion of YTHDF1 by enhancing binding with Crm1, thus upregulating downstream target (e.g. c-Myc) expression. Molecular dynamics simulations suggest that O-GlcNAcylation at S197 promotes the binding between the nuclear export signal motif and Crm1 through increasing hydrogen bonding. Mouse xenograft assays further demonstrate that YTHDF1-AFA mutants decreased the colon cancer mass and size via decreasing c-Myc expression. In sum, we found that YTHDF1 is a nucleocytoplasmic protein, whose cytosolic localization is dependent on O-GlcNAc modification. We propose that the OGT-YTHDF1-c-Myc axis underlies colorectal cancer tumorigenesis.

Keywords:  Crm1; O-GlcNAc; YTHDF1; c-Myc; m(6)A

DOI:  https://doi.org/10.1016/j.jbc.2023.104738
eNeuro. 2023 Apr 24. pii: ENEURO.0338-22.2023. [Epub ahead of print]

Upregulation of UBR1 m6A methylation by METTL14 inhibits autophagy in spinal cord injury.

Changsheng Wang, Xitian Zhu, Rongsheng Chen, Xiaobo Zhang, Nancheng Lian.

  Objective: Gene Expression Omnibus database shows significantly downregulated expression of ubiquitin protein ligase E3 component N-recognin 1 (UBR1) in spinal cord injury (SCI). In this study, we investigated the mechanism of action of UBR1 in SCI.Methods: Following the establishment of SCI models in rats and PC12 cells, Basso-Beattie-Bresnahan score and H&E and Nissl staining were used to evaluate SCI. The localization of NeuN/LC3 and the expression of LC3II/I, Beclin-1, and p62 were detected to assess autophagy. The expression of Bax, Bcl-2, and cleaved caspase-3 was detected and TdT-mediated dUTP-biotin nick end-labelling staining was employed to determine the changes in apoptosis. The N(6)-methyladenosine (m6A) modification level of UBR1 was analyzed by methylated RNA immunoprecipitation, and the binding of METTL14 and UBR1 mRNA was analysed by photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation.Results: UBR1 was poorly expressed, and METTL14 was highly expressed in rat and cell models of SCI. UBR1 overexpression or METTL14 knockdown enhanced motor function in rats with SCI. Moreover, this modification increased Nissl bodies and autophagy and inhibited apoptosis in the spinal cord of SCI rats. METTL14 silencing reduced the m6A modification level of UBR1 and enhanced UBR1 expression. Importantly, UBR1 knockdown nullified METTL14 knockdown -induced autophagy promotion and apoptosis reduction.Conclusion: The METTL14-catalyzed m6A methylation of UBR1 promoted apoptosis and inhibited autophagy in SCI.

Keywords:  METTL14; UBR1; apoptosis; autophagy; m6A methylation; spinal cord injury

DOI:  https://doi.org/10.1523/ENEURO.0338-22.2023
Int J Mol Sci. 2023 Apr 07. pii: 6905. [Epub ahead of print]24(8):

YTHDF1 Promotes Bladder Cancer Cell Proliferation via the METTL3/YTHDF1-RPN2-PI3K/AKT/mTOR Axis.

Junlong Zhu, Hang Tong, Yan Sun, Tinghao Li, Guang Yang, Weiyang He.

  N6-methyladenosine (m6A) is the most common mRNA modification and it plays a critical role in tumor progression, prognoses and therapeutic response. In recent years, more and more studies have shown that m6A modifications play an important role in bladder carcinogenesis and development. However, the regulatory mechanisms of m6A modifications are complex. Whether the m6A reading protein YTHDF1 is involved in the development of bladder cancer remains to be elucidated. The aims of this study were to determine the association between METTL3/YTHDF1 and bladder cancer cell proliferation and cisplatin resistance to explore the downstream target genes of METTL3/YTHDF1 and to explore the therapeutic implications for bladder cancer patients. The results showed that the reduced expression of METTL3/YTHDF1 could lead to decreased bladder cancer cell proliferation and cisplatin sensitivity. Meanwhile, overexpression of the downstream target gene, RPN2, could rescue the effect of reduced METTL3/YTHDF1 expression on bladder cancer cells. In conclusion, this study proposes a novel METTL3/YTHDF1-RPN2-PI3K/AKT/mTOR regulatory axis that affects bladder cancer cell proliferation and cisplatin sensitivity.

Keywords:  N6-methyladenosine; bladder cancer; cell proliferation; drug sensitivity; m6A

DOI:  https://doi.org/10.3390/ijms24086905
Sci Rep. 2023 Apr 24. 13(1): 6617

Characterization of the m6A regulator-mediated methylation modification patterns in oral squamous cell carcinoma.

Lu Pan, He She, Keyi Wang, Wenhui Xia, Haonan Tang, Yuan Fan, Jinhai Ye.

N6-methyladenosine (m6A) is a form of posttranscriptional modification that plays important roles in cancer including oral squamous cell carcinoma (OSCC). Most studies to date have focused on a limited number of regulators and oncogenic pathways, thus failing to provide comprehensive insight into the dynamic effects of m6A modification. In addition, the role of m6A modification in shaping immune cell infiltration in OSCC has yet to be clarified. This study was designed to assess m6A modification dynamics in OSCC and to understand how such modifications influence clinical immunotherapeutic treatment outcomes. m6A modification patterns linked with 23 m6A regulators were analyzed in 437 OSCC patients from TCGA and GEO cohorts. These patterns were then quantified through m6A score based on algorithms derived from a principal component analysis (PCA) approach. The m6A modification patterns of OSCC samples were grouped into two clusters based on the m6A regulators expression, and immune cell infiltration was linked with the 5-year survival outcomes of patients in these clusters. 1575 genes associated with OSCC patient prognosis were identified and used to re-cluster these samples into two groups. Patients in clusters exhibiting higher levels of m6A regulator expression exhibited poorer overall survival (OS), whereas patients with high m6A scores survived for longer (p < 0.001). The overall mortality rates in the groups of patients with low and high m6A scores were 55% and 40%, respectively, and the m6A score distributions in clusters of patients grouped by m6A modification patterns and gene expression further supported the link between a high m6A score and better prognostic outcomes. Immunophenoscore (IPS) values for patients in different m6A score groups suggested that the use of PD-1-specific antibodies or CTLA-4 inhibitors alone or in combination would yield superior treatment outcomes in patients in the high-m6A score group relative to the low-m6A score group. m6A modification patterns are relevant to heterogeneity in OSCC. Detailed analyses of m6A modification patterns may thus offer novel insight regarding immune cell infiltration within the OSCC tumor microenvironment, guiding novel efforts to provide patients with more effective immunotherapeutic interventions.

DOI: https://doi.org/10.1038/s41598-023-33891-9
J Clin Med. 2023 Apr 14. pii: 2863. [Epub ahead of print]12(8):

Alteration of m6A-Tagged RNA Profiles in Bone Originated from Periprosthetic Joint Infection.

Yuanqing Cai, Xiaoqing Chen, Changyu Huang, Yang Chen, Chaofan Zhang, Zida Huang, Wenming Zhang, Yusen Tang, Xinyu Fang.

  Periprosthetic joint infection (PJI) is a devastating complication. This study aimed to unravel the veil of the N6-methyladenine (m6A) modification in PJI. Synovium, synovial fluid, sonication fluid and bone samples were collected intraoperatively from Staphylococcus aureus PJI and aseptic failure (AF) patients. The overall m6A level was detected by the m6A RNA methylation quantification kit, and the expression of m6A-related genes was quantified by real-time PCR and Western blot. Finally, an epitranscriptomic microarray and bioinformatics analysis were performed. We showed that there was a significant difference in overall m6A level between the PJI group and the AF group (PJI group had a higher overall m6A level). The expression level of METTL3 was higher in the PJI group than that in the AF group. There were 2802 differential m6A-modified mRNAs. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that differential m6A-modified mRNAs were significantly enriched in the NOD-like receptor signaling pathway, Th17 cell differentiation and the IL-17 signaling pathway, which indicates that the m6A modification might be involved in the processes of infection and immune response, bone metabolism and programmed cell death in PJI. In summary, the present work demonstrated that m6A modification plays a role in PJI and might be a therapeutic target for developing effective treatment strategies.

Keywords:  N6-methyladenine; Staphylococcus aureus; mRNA; periprosthetic joint infection; profiles

DOI:  https://doi.org/10.3390/jcm12082863
Nat Aging. 2023 Apr 06.

m6A epitranscriptomic regulation of tissue homeostasis during primate aging.

Zeming Wu, Mingming Lu, Di Liu, Yue Shi, Jie Ren, Si Wang, Ying Jing, Sheng Zhang, Qian Zhao, Hongyu Li, Zihui Yu, Zunpeng Liu, Shijia Bi, Tuo Wei, Yun-Gui Yang, Jingfa Xiao, Juan Carlos Izpisua Belmonte, Jing Qu, Weiqi Zhang, Weimin Ci, Guang-Hui Liu.

How N6-methyladenosine (m6A), the most abundant mRNA modification, contributes to primate tissue homeostasis and physiological aging remains elusive. Here, we characterize the m6A epitranscriptome across the liver, heart and skeletal muscle in young and old nonhuman primates. Our data reveal a positive correlation between m6A modifications and gene expression homeostasis across tissues as well as tissue-type-specific aging-associated m6A dynamics. Among these tissues, skeletal muscle is the most susceptible to m6A loss in aging and shows a reduction in the m6A methyltransferase METTL3. We further show that METTL3 deficiency in human pluripotent stem cell-derived myotubes leads to senescence and apoptosis, and identify NPNT as a key element downstream of METTL3 involved in myotube homeostasis, whose expression and m6A levels are both decreased in senescent myotubes. Our study provides a resource for elucidating m6A-mediated mechanisms of tissue aging and reveals a METTL3-m6A-NPNT axis counteracting aging-associated skeletal muscle degeneration.

DOI: https://doi.org/10.1038/s43587-023-00393-2
Cell Cycle. 2023 Apr 23. 1-14

METTL3 affects FLT3-ITD+ acute myeloid leukemia by mediating autophagy by regulating PSMA3-AS1 stability.

Shenghao Wu, Shanshan Weng, Wenjin Zhou, Yuemiao Chen, Zhen Liu.

  The study was designed to explore the role of PSMA3-AS1 in initiation and progression of acute myeloid leukemia (AML) and investigate its action mechanism. Expression of PSMA3-AS1, miR-20a-5p and ATG16L1 both in vitro and in vivo was measured by qRT-PCR. The expression of protein was detected by western blot assay. Edu staining and flow cytometry were utilized to measure cell proliferation and apoptosis. Potential target was predicted by bioinformatics and was verified by dual-luciferase report gene assay and RNA pull down assay. QRT-PCR was used to quantify autophagy (LC3, Beclin1, P62) related genes. The m6A modification test is used to verify the effect of METTL3 on PSMA3-AS1. Tumor model was used to identify the effect of PSMA3-AS1 on tumor growth in vivo, and immunohistochemistry was applied to detect expression of ki67 and TUNEL. The results indicate that PSMA3-AS1 was upregulated in FLT3-ITD+ AML patients. Si-PSMA3-AS1 could inhibit the proliferation, autophagy and promote the apoptosis in MV4-11 and Molm13 cells. METTL3 could enhance the PSMA3-AS1 RNA stability. In addition, this study revealed that PSMA3-AS1 affected FLT3-ITD+ AML by targeting expression of miR-20a-5p, and miR-20a-5p further modulated expression of ATG16L1, an mRNA that down-regulated in AML, to affect disease advancement. PSMA3-AS1 could promote FLT3-ITD+ AML progression by regulating the level of autophagy through miR-20a-5p/ATG16L1 pathway. In addition, the increase of PSMA3-AS1 may be caused by the involvement of METTL3 in regulating its stability. This discovery will provide new horizons for early screening and targeted therapy of FLT3-ITD+ AML.

Keywords:  Acute myeloid leukemia (AML); FLT3-ITD; N6-methyladenosine; PSMA3-AS1; autophagy

DOI:  https://doi.org/10.1080/15384101.2023.2204770
Epigenetics. 2023 Dec;18(1): 2204772

m6A-modification regulated circ-CCT3 acts as the sponge of miR-378a-3p to promote hepatocellular carcinoma progression.

Hua Liu, Yifan Jiang, Jiahua Lu, Chuanhui Peng, Zhenan Ling, Yunhao Chen, Diyu Chen, Rongliang Tong, Shusen Zheng, Jian Wu.

  Background: Circular RNA (circRNA) plays a critical role in tumour progression. Circ-CCT3, a particularly abundant circRNA, was proposed to be involved in tumorigenesis. However, the role of circ-CCT3 in hepatocellular carcinoma remains elusive.Methods: Here, circ-CCT3 (a circRNA derived from exons 3, 4 and 5 of the CCT3 gene, hsa_circ_0004680) was identified by circRNA microarray and validated by qRT-PCR. RNA immunoprecipitation (RIP) was performed to confirm the binding between ALKBH5 along with METTL3 and circ-CCT3. Methylated RNA Immunoprecipitation (MeRIP) was used to detect the N6-methyladenosine (m 2A) levels of circ-CCT3. CircRNAs in vivo precipitation, luciferase reporter assay, biotin-coupled microRNA capture, and fluorescence in situ hybridization were conducted to assess the interaction between circ-CCT3 and miR-378a-3p. The functions of circ-CCT3 in HCC were evaluated both in vitro and in vivo.Results: We demonstrated that circ-CCT3 was highly expressed in HCC which indicated the poor prognosis. Circ-CCT3 expression served as an independent risk factor for overall survival in patients with HCC. Knocking-down of circ-CCT3 inhibited the proliferation, invasion and migration of HCC cells, and angiogenesis of HUVEC. Mechanistically, ALKBH5 and METTL3 could bind and regulate m A-modification of circ-CCT3. Further, circ-CCT3 upregulated the expression of FLT-1 by sponging miR-378a-3p.Conclusions: Circ-CCT3 was significantly up-regulated in HCC and promoted liver cancer development via miR-378a-3p-FLT1 axis. It was also found that circ-CCT3 was under m A-modification mediated by ALKBH5 and METTL3. Our study highlights circ-CCT3 as a potential therapeutic target of HCC treatment, which provides a novel understanding on mechanisms of circRNAs in HCC progression.

Keywords:  Circ-CCT3; FLT1; Hepatocellular carcinoma; MiR-378a-3p; N6-methyladenosine

DOI:  https://doi.org/10.1080/15592294.2023.2204772
Hepatology. 2023 Apr 26.

Purine anabolism creates therapeutic vulnerability in hepatocellular carcinoma via m6A-mediated epitranscriptomic regulation.

Man Hsin Hung, Ching Wen Chang, Kathy Cheng Wang, Jittiporn Chaisaingmongkol, Mathuros Ruchirawat, Tim F Greten, Xin Wei Wang.

BACKGROUND AND AIMS: Purines are building blocks for cellular genome and excessive purine nucleotides are seen in tumors. However, how purine metabolism is dysregulated in tumor and impacting tumorigenesis remains elusive.
APPROACH AND RESULTS: Transcriptomic and metabolomic analysis of purine biosynthesis and purine degradation pathways were performed in the tumor and associated non-tumor liver tissues obtained from 62 patients with hepatocellular carcinoma (HCC), one of the most lethal cancers worldwide. We found that most genes in purine synthesis are upregulated while genes in purine degradation are inhibited in HCC tumors. High purine anabolism is associated with unique somatic mutational signatures linked to patient prognosis. Mechanistically, we discover that increasing purine anabolism promotes epitranscriptomic dysregulation of DDR machinery through upregulating RNA N6-methyladenosine modification. High purine anabolic HCC is sensitive to DDR-targeting agents but not to standard HCC treatments, correlating with the clinical outcomes in five independent HCC cohorts containing 724 patients. We further showed that high purine anabolism determines the sensitivity to DDR-targeting agents in 5 HCC cell lines in vitro and in vivo.
CONCLUSION: Our results reveal a central role of purine anabolism in regulating DDR, which could be therapeutically exploited in HCC.

DOI: https://doi.org/10.1097/HEP.0000000000000420
Biol Direct. 2023 Apr 23. 18(1): 19

IGF2BP2 promotes colorectal cancer progression by upregulating the expression of TFRC and enhancing iron metabolism.

Tian-Yue Liu, Chen-Chen Hu, Chen-Ying Han, Si-Yi Mao, Wen-Xin Zhang, Yi-Ming Xu, Yuan-Jie Sun, Dong-Bo Jiang, Xi-Yang Zhang, Jia-Xing Zhang, Jing Wang, Xu-Peng Qiao, Jing-Yu Pan, Shu-Ya Yang, Kun Yang.

   BACKGROUND: Colorectal cancer (CRC) is one of the most common malignant tumors of the digestive system, ranking third for morbidity and mortality worldwide. At present, no effective control method is available for this cancer type. In tumor cells, especially iron metabolization, is necessary for its growth and proliferation. High levels of iron are an important feature to maintain tumor growth; however, the overall mechanism remains unclear.
METHODS: We used western blotting, immunohistochemistry (IHC) and real-time quantitative PCR to analyze the expression of IGF2BP2 in cell lines and tissues. Further, RNA-sequencing, RNA immunoprecipitation and methylated RNA immunoprecipitation experiments explored the specific binding of target genes. Moreover, the RNA stability assay was performed to determine the half-life of genes downstream of IGF2BP2. In addition, the Cell Counting Kit-8, colony formation assay, 5-ethynyl-2'-deoxyuridine assay and flow cytometry were used to evaluate the effects of IGF2BP2 on proliferation and iron metabolism. Lastly, the role of IGF2BP2 in promoting CRC growth was demonstrated in animal models.
RESULTS: We observed that IGF2BP2 is associated with iron homeostasis and that TFRC is a downstream target of IGF2BP2. Further, overexpression of TFRC can rescue the growth of IGF2BP2-knockdown CRC cells. Mechanistically, we determined that IGF2BP2 regulates TFRC methylation via METTL4, thereby regulating iron metabolism and promoting CRC growth. Furthermore, using animal models, we observed that IGF2BP2 promotes CRC growth.
CONCLUSION: IGF2BP2 regulates TFRC mRNA methylation via METTL4, thereby regulating iron metabolism and promoting CRC growth. Our study highlights the key roles of IGF2BP2 in CRC carcinogenesis and the iron transport pathways.

Keywords:  Colorectal cancer; IGF2BP2; Iron metabolism; m6A-TFRC

DOI:  https://doi.org/10.1186/s13062-023-00373-x
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2023 Apr;39(4): 303-310

[Knockdown of IGF2BP2 inhibits colorectal cancer cell proliferation, migration and promotes tumor immunity by down-regulating MYC expression].

Tianyue Liu, Chenying Han, Chenchen Hu, Siyi Mao, Yuanjie Sun, Shuya Yang, Kun Yang.

Objective To investigate the effect of insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2) on the proliferation, migration and tumor immune microenvironment of colorectal cancer cells and its possible molecular mechanism. Methods The Cancer Genome Atlas (TCGA) database was used to analyze the expression levels of IGF2BP2 and MYC in colorectal cancer and adjacent tissues. The expression of IGF2BP2 in HCT-116 and SW480 human colorectal cancer cells was silenced by RNA interference (RNAi), and the silencing effect was detected by quantitative real-time PCR. After knocking down IGF2BP2, colony formation assay, CCK-8 assay and 5-ethynyl-2'-deoxyuridine (EdU) assay were employed to detect cell colony formation and proliferation ability. TranswellTM assay was used to detect cell migration ability. Quantitative real-time PCR was used to detect the mRNA expression of IGF2BP2, MYC, tumor necrosis factor-α (TNF-α), transforming growth factor-β (TGF-β) and interleukin-10 (IL-10). The protein expression of IGF2BP2 and MYC was detected by western blot. The binding ability of IGF2BP2 and MYC in HCT-116 cells was detected by quantitative real-time PCR after RNA immunoprecipitation. Results The results of TCGA database showed that the expression of IGF2BP2 and MYC in colorectal cancer tissues was significantly higher than that in adjacent tissues, and the survival time of colorectal cancer patients with high expression of IGF2BP2 was shorter. After silencing IGF2BP2, the viability, proliferation and migration of HCT-116 and SW480 cells were decreased. The mRNA expression of MYC, TGF-β and IL-10 in IGF2BP2 knockdown group was significantly decreased, while the expression of TNF-α mRNA was increased. The expression of MYC protein and the stability of MYC mRNA were significantly decreased. RIP-qPCR results showed that IGF2BP2 could bind to MYC mRNA. Conclusion Knockdown of IGF2BP2 inhibits colorectal cancer cell proliferation, migration and promotes tumor immunity by down-regulating MYC expression.
Eur J Pharmacol. 2023 Apr 26. pii: S0014-2999(23)00247-9. [Epub ahead of print] 175736

5-Azacytidine promotes HCC cell metastasis by up-regulating RDH16 expression.

Li Le, Wu Qipeng, Miao Chunmeng, Muhammad Hasnat, Zhang Luyong, Jiang Zhenzhou, Yu Qinwei.

  The level of DNA methylation could affect the expression of tumor promoting and tumor suppressor genes. DNA methyltransferase inhibitors could reduce high methylation levels in cancer and inhibit the progression of a variety of cancers, including HCC. However, the pro-metastatic effect of DNA methyltransferase inhibitors in some cancers suggest the potential risk of their use. Whether DNA methyltransferase inhibitors also promote metastasis in HCC remains unclear. Our study will explore the effect of DNA methyltransferase inhibitor 5-Azacytidine on HCC metastasis. Our study found that 5-Azacytidine inhibited the proliferation of HCC cells while promoting in vitro and in vivo metastasis of HCC. Mechanistically, our study showed that 5-Azacytidine increased the expression of RDH16 by decreasing the methylation of RDH16 gene promoter. RDH16 is a highly methylated gene and its expression is very low in hepatocellular carcinoma. 5-Azacytidine promoted the migration of hepatocellular carcinoma cells by increasing the expression of RDH16. Our results suggest that 5-Azacytidine up-regulates the expression of RDH16 by decreasing the methylation level of RDH16, and then promoting HCC metastasis. These findings suggest that 5-Azacytidine and even other DNA methyltransferase inhibitors may have the risk of promoting metastasis in HCC treatment. RDH16 could be used as a pro-metastasis biomarker in the treatment of HCC with DNA methyltransferase inhibitors.

Keywords:  5-Azacytidine; DNA methylation; Migration; RDH16

DOI:  https://doi.org/10.1016/j.ejphar.2023.175736
J Transl Med. 2023 Apr 23. 21(1): 276

The activation of mTOR signalling modulates DNA methylation by enhancing DNMT1 translation in hepatocellular carcinoma.

Mengke Chen, Yi Fang, Meinong Liang, Ning Zhang, Xinyue Zhang, Lixia Xu, Xuxin Ren, Qingfeng Zhang, Yufeng Zhou, Sui Peng, Jun Yu, Judeng Zeng, Xiaoxing Li.

   BACKGROUND: Both dysregulation of mechanistic target of rapamycin (mTOR) signalling and DNA methylation patterns have been shown to be closely associated with tumor progression and serve as promising targets for hepatocellular carcinoma (HCC) therapy. Although their respective roles in HCC have been extensively revealed, the existence of molecular interactions between them remains largely unknown.
METHODS: The association of DNA methylation and mTOR signalling in HCC tissues and cell lines was assessed. A Kaplan‒Meier analysis was applied to estimate the overall survival (OS) and recurrence-free survival (RFS) of HCC patients. The modulation of DNMT1 by mTOR in HCC cell lines was determined. The effect of the drug combination in cell lines and mouse models was examined.
RESULTS: The results showed that the DNA methylation level was positively associated with the activation of mTOR signalling in HCC tissues and cell lines. Moreover, HCC patients with higher DNA methylation levels and enhanced activation of mTOR signalling exhibited the worst prognosis. Then, we screened methylation-related enzymes and found that the activation of mTOR signalling increased DNMT1 expression and activity. In addition, mTOR enhanced the translational efficiency of DNMT1 in a 4E-BP1-dependent manner, which is based on the pyrimidine rich translational element (PRTE)-containing 5'UTR of DNMT1. Moreover, we demonstrated that the combined inhibition of mTOR and DNMT synergistically inhibited HCC growth in vitro and in vivo.
CONCLUSIONS: In addition to some already identified pro-cancer downstream molecules, the activation of mTOR signalling was found to promote DNA methylation by increasing the translation of DNMT1. Furthermore, combined targeting of mTOR and DNMT1 has been demonstrated to have a more effective tumor suppressive function in HCC.

Keywords:  DNA methylation; DNMT1; Hepatocellular carcinoma; Protein translation; Pyrimidine rich translational element

DOI:  https://doi.org/10.1186/s12967-023-04103-9
JCI Insight. 2023 Apr 24. pii: e164178. [Epub ahead of print]8(8):

MKRN3 inhibits puberty onset via interaction with IGF2BP1 and regulation of hypothalamic plasticity.

Lydie Naulé, Alessandra Mancini, Sidney A Pereira, Brandon M Gassaway, John R Lydeard, John C Magnotto, Han Kyeol Kim, Joy Liang, Cynara Matos, Steven P Gygi, Florian T Merkle, Rona S Carroll, Ana Paula Abreu, Ursula B Kaiser.

  Makorin ring finger protein 3 (MKRN3) was identified as an inhibitor of puberty initiation with the report of loss-of-function mutations in association with central precocious puberty. Consistent with this inhibitory role, a prepubertal decrease in Mkrn3 expression was observed in the mouse hypothalamus. Here, we investigated the mechanisms of action of MKRN3 in the central regulation of puberty onset. We showed that MKRN3 deletion in hypothalamic neurons derived from human induced pluripotent stem cells was associated with significant changes in expression of genes controlling hypothalamic development and plasticity. Mkrn3 deletion in a mouse model led to early puberty onset in female mice. We found that Mkrn3 deletion increased the number of dendritic spines in the arcuate nucleus but did not alter the morphology of GnRH neurons during postnatal development. In addition, we identified neurokinin B (NKB) as an Mkrn3 target. Using proteomics, we identified insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) as another target of MKRN3. Interactome analysis revealed that IGF2BP1 interacted with MKRN3, along with several members of the polyadenylate-binding protein family. Our data show that one of the mechanisms by which MKRN3 inhibits pubertal initiation is through regulation of prepubertal hypothalamic development and plasticity, as well as through effects on NKB and IGF2BP1.

Keywords:  Endocrinology; Mouse models; Neurodevelopment; Neuroendocrine regulation; Neuroscience

DOI:  https://doi.org/10.1172/jci.insight.164178