bims-rimeca 2023-04-09 papers

bims-rimeca

Biomed News

on RNA methylation in cancer

Issue of 2023–04–09
thirty-six papers selected by
Sk Ramiz Islam, Saha Institute of Nuclear Physics

Targeted m6A demethylation of ITGA6 mRNA by a multisite dCasRx-m6A editor inhibits bladder cancer development.
N6-methyladenosine (m6A) modification of ribosomal RNAs (rRNAs): Critical roles in mRNA translation and diseases.
FTO-mediated m6A demethylation of pri-miR-3591 alleviates osteoarthritis progression.
m6A reader IGF2BP1 accelerates apoptosis of high glucose-induced vascular endothelial cells in a m6A-HMGB1 dependent manner.
The role of RNA methyltransferase METTL3 in gynecologic cancers: Results and mechanisms.
m6A methyltransferase METTL3 programs CD4+ T-cell activation and effector T-cell differentiation in systemic lupus erythematosus.
Comprehensive analysis of m6A RNA methylation modification patterns and the immune microenvironment in osteoarthritis.
VIRMA Promotes Nasopharyngeal Carcinoma Tumorigenesis and Metastasis by Upregulation of E2F7 in an m6A-Dependent Manner.
WTAP-Mediated m6A RNA Methylation Regulates the Differentiation of Bone Marrow Mesenchymal Stem Cells via the miR-29b-3p/HDAC4 Axis.
N6-methyladenosine reader YTHDF2 promotes multiple myeloma cell proliferation through EGR1/p21cip1/waf1/CDK2-Cyclin E1 axis-mediated cell cycle transition.
Chinese Ecliptae herba (Eclipta prostrata (L.) L.) extract and its component wedelolactone enhances osteoblastogenesis of bone marrow mesenchymal stem cells via targeting METTL3-mediated m6A RNA methylation.
WTAP promotes macrophage recruitment and increases VEGF secretion via N6-methyladenosine modification in corneal neovascularization.
Comprehensive analyses of molecular features, prognostic values, and regulatory functionalities of m6A-modified long non-coding RNAs in lung adenocarcinoma.
Role of N6-adenosine-methyltransferase subunits METTL3 and METTL14 in the biological properties of periodontal ligament cells.
Profiling of N6-methyladenosine methylation in porcine longissimus dorsi muscle and unravelling the hub gene ADIPOQ promotes adipogenesis in an m6A-YTHDF1-dependent manner.
Aberrant RNA m6A modification in gastrointestinal malignancies: versatile regulators of cancer hallmarks and novel therapeutic opportunities.
The m6A reader YTHDC1 and the RNA helicase DDX5 control the production of rhabdomyosarcoma-enriched circRNAs.
Reading and writing of mRNA m6A modification orchestrate maternal-to-zygotic transition in mice.
Single-cell mapping of N6-methyladenosine in esophageal squamous cell carcinoma and exploration of the risk model for immune infiltration.
METTL3-mediated downregulation of splicing factor SRSF11 is associated with carcinogenesis and poor survival of cancer patients.
When N7-methyladenosine modification meets cancer: Emerging frontiers and promising therapeutic opportunities.
Development a m6A regulators characterized by the immune cell infiltration in stomach adenocarcinoma for predicting the prognosis and immunotherapy response.
Expression patterns of eight RNA-modified regulators correlating with immune infiltrates during the progression of osteoarthritis.
FTO regulates the DNA damage response via effects on cell-cycle progression.
METTL14 Promotes Oral Squamous Cell Carcinoma Progression by Regulating the mRNA and m6A Levels of CALD1.
N6-methyladenosine modifications in maternal-fetal crosstalk and gestational diseases.
PRMT3-mediated arginine methylation of IGF2BP1 promotes oxaliplatin resistance in liver cancer.
Analysis of eight types of RNA modification regulators and their correlation with the prognosis in hepatocellular carcinoma.
Contribution of A-to-I RNA editing, M6A RNA Methylation, and Alternative Splicing to physiological brain aging and neurodegenerative diseases.
Cuproptosis illustrates tumor micro-environment features and predicts prostate cancer therapeutic sensitivity and prognosis.
Systematic comparison of tools used for m6A mapping from nanopore direct RNA sequencing.
Phase separation of DDX21 promotes colorectal cancer metastasis via MCM5-dependent EMT pathway.
RUNX2 Reverses p53-Induced Chemotherapy Resistance in Gastric Cancer.
Characterization of heterogeneous metabolism in hepatocellular carcinoma identifies new therapeutic target and treatment strategy.
IGFBP-3 promotes cachexia-associated lipid loss by suppressing insulin-like growth factor/insulin signaling.
IGFBP‑rP1 affects the proliferation, apoptosis and macrophage polarization of endometrial cancer cells by regulating the PI3K/AKT pathway.

J Adv Res. 2023 Mar 30. pii: S2090-1232(23)00092-9. [Epub ahead of print]

Targeted m6A demethylation of ITGA6 mRNA by a multisite dCasRx-m6A editor inhibits bladder cancer development.

Xiaoling Ying, Yapeng Huang, Bixia Liu, WenYu Hu, Ding Ji, Cong Chen, Haiqing Zhang, Yaomin Liang, Yifan Lv, Weidong Ji.

   INTRODUCTION: N6-methyladenosine (m6A) modification contributes to the pathogenesis and development of various cancers, including bladder cancer (BCa). In particular, integrin α6 (ITGA6) promotes BCa progression by cooperatively regulating multisite m6A modification. However, the therapeutic effect of targeting ITGA6 multisite m6A modifications in BCa remains unknown.
OBJECTIVES: We aim to develop a multisite dCasRx- m6A editor for assessing the effects of the multisite dCasRx-m6A editor targeted m6A demethylation of ITGA6 mRNA in BC growth and progression.
METHODS: The multisite dCasRx- m6A editor was generated by cloning. m6A-methylated RNA immunoprecipitation (meRIP), luciferase reporter, a single-base T3 ligase-based qPCR-amplification, Polysome profiling and meRIP-seq experiments were performed to determine the targeting specificity of the multisite dCasRx-m6A editor. We performed cell phenotype analysis and used in vivo mouse xenograft models to assess the effects of the multisite dCasRx-m6A editor in BC growth and progression.
RESULTS: We designed a targeted ITGA6 multi-locus guide (g)RNA and established a bidirectional deactivated RfxCas13d (dCasRx)-based m6A-editing platform, comprising a nucleus-localized dCasRx fused with the catalytic domains of methyltransferase-like 3 (METTL3-CD) or α-ketoglutarate-dependent dioxygenase alkB homolog 5 (ALKBH5-CD), to simultaneously manipulate the methylation of ITGA6 mRNA at four m6A sites. The results confirmed the dCasRx-m6A editor modified m6A at multiple sites in ITGA6 mRNA, with low off-target effects. Moreover, targeted m6A demethylation of ITGA6 mRNA by the multisite dCasRx-m6A editor significantly reduced BCa cell proliferation and migration in vitro and in vivo. Furthermore, the dCasRx-ALKBH5-CD and ITGA6 multi-site gRNA delivered to 5-week-old BALB/cJNju-Foxn1nu/Nju nude mice via adeno-associated viral vectors significantly inhibited BCa cell growth.
CONCLUSION: Our study proposes a novel therapeutic tool for the treatment of BC by applying the multisite dCasRx-m6A editor while highlighting its potential efficacy for treating other diseases associated with abnormal m6A modifications.

Keywords:  Adeno-associated viral; Bladder cancer; ITGA6; Multisite; N(6)-methyladenosine; dCasRx

DOI:  https://doi.org/10.1016/j.jare.2023.03.010
Genes Dis. 2023 Jan;10(1): 126-134

N6-methyladenosine (m6A) modification of ribosomal RNAs (rRNAs): Critical roles in mRNA translation and diseases.

Kexin Lei, Shuibin Lin, Quan Yuan.

  As key components of the ribosome and the most abundant RNA species, the rRNAs are modified during ribosome formation. N6-methyladenosine (m6A) is a conserved RNA modification occurring on different RNA species including rRNAs. Recently, it has been reported that ZCCHC4 and METTL5 are methyltransferases that mediate m6A modification of human 28S and 18S rRNA, respectively. The newly discovered biological functions of the two methyltransferases include regulation of mRNA translation, cell proliferation, cell differentiation, stress response, and other biological processes. Both of them, especially METTL5, have been proved to be associated with a variety of diseases such as intellectual disability, cancer, congenital dysplasia and have potential clinical application as biomarkers and therapeutic targets.

Keywords:  Biological processes; Diseases; METTL5; ZCCHC4; m6A

DOI:  https://doi.org/10.1016/j.gendis.2021.10.005
Arthritis Res Ther. 2023 04 01. 25(1): 53

FTO-mediated m6A demethylation of pri-miR-3591 alleviates osteoarthritis progression.

Wengang Liu, Tao Jiang, Wei Zheng, Jiayuan Zhang, Anan Li, Chao Lu, Zhaowei Lin.

   OBJECTIVES: Increasing evidence have demonstrated the N6-methyladenosine (m6A) plays critical roles in osteoarthritis (OA) progression, but the role of m6A in OA has not been completely illuminated. Herein, we investigated the function and underlying mechanism of m6A demethylase fat mass and obesity-associated protein (FTO) in OA progression.
MATERIALS AND METHODS: The FTO expression was detected in mice OA cartilage tissues and lipopolysaccharide (LPS)-stimulated chondrocytes. Gain-of-function assays was used to evaluate the role of FTO in OA cartilage injury in vitro and in vivo. The miRNA-sequencing, RNA-binding protein immunoprecipitation (RIP), luciferase reporter assay, and in vitro pri-miRNA processing assays were conducted to confirm that FTO modulated the pri-miR-3591 process in an m6A-dependent manner and then the binding sites of miR-3591-5p with PRKAA2.
RESULTS: FTO was outstandingly downregulated in LPS-stimulated chondrocytes and OA cartilage tissues. FTO overexpression enhanced the proliferation, suppressed apoptosis, and decreased degradation of extracellular matrix in LPS-induced chondrocytes, whereas FTO knockdown contributed to the opposite effects. In vivo animal experiments showed that FTO overexpression markedly alleviated OA mice cartilage injury. Mechanically, FTO-mediated m6A demethylation of pri-miR-3591 leaded to a maturation block of miR-3591-5p, which relieved the inhibitory effect of miR-3591-5p on PRKAA2 and then promoted the increase of PRKAA2, thereby alleviating OA cartilage damage.
CONCLUSIONS: Our results attested that FTO alleviated the OA cartilage damage by mediating FTO/miR-3591-5p/PRKAA2 axis, which provided fresh insights into the therapeutic strategies for OA.

Keywords:  FTO; N6-methyladenosine (m6A); Osteoarthritis; PRKAA2; miR-3591-5p

DOI:  https://doi.org/10.1186/s13075-023-03035-5
PeerJ. 2023 ;11 e14954

m6A reader IGF2BP1 accelerates apoptosis of high glucose-induced vascular endothelial cells in a m6A-HMGB1 dependent manner.

Anru Liang, Jianyu Liu, Yanlin Wei, Yuan Liao, Fangxiao Wu, Jiang Ruan, Junjun Li.

  Emerging evidence indicates that N6-methyladenosine (m6A) plays a critical role in vascular biological characteristic. In diabetes mellitus pathophysiology, high glucose (HG)-induced vascular endothelial dysfunction is associated with diabetes vascular complications. Nevertheless, the underlying mechanism of high glucose (HG)-related m6A regulation on vascular endothelial cells is still unclear. Results indicated that m6A reader insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) was up-regulated in HG-treated human umbilical vascular endothelium cells (HUVECs) comparing to normal group. Functionally, results indicated that IGF2BP1 knockdown recovered the proliferation of HUVECs inhibited by HG-administration. Besides, IGF2BP1 knockdown reduced the apoptosis induced by HG-administration. Mechanistically, IGF2BP1 interacted with HMGB1 mRNA and stabilized its expression of m6A-modified RNA. Therefore, these findings provided compelling evidence demonstrating that m6A reader IGF2BP1 contributes to the proliferation and apoptosis of vascular endothelial cells in hyperglycaemia, serving as a target for development of diabetic angiopathy therapeutics.

Keywords:  IGF2BP1; N6-methyladenosine; Vascular endothelial cells

DOI:  https://doi.org/10.7717/peerj.14954
Front Pharmacol. 2023 ;14 1156629

The role of RNA methyltransferase METTL3 in gynecologic cancers: Results and mechanisms.

Yuxiang Zhang, Na Zhang.

  N6-methyladenosine (m6A) methylation is the most prevalent mRNA modification in eukaryotes, and it is defined as the methylation of nitrogen atoms on the six adenine (A) bases of RNA in the presence of methyltransferases. Methyltransferase-like 3 (Mettl3), one of the components of m6A methyltransferase, plays a decisive catalytic role in m6A methylation. Recent studies have confirmed that m6A is associated with a wide spectrum of biological processes and it significantly affects disease progression and prognosis of patients with gynecologic tumors, in which the role of Mettl3 cannot be ignored. Mettl3 is involved in numerous pathophysiological functions, such as embryonic development, fat accumulation, and tumor progression. Moreover, Mettl3 may serve as a potential target for treating gynecologic malignancies, thus, it may benefit the patients and prolong survival. However, there is a need to further study the role and mechanism of Mettl3 in gynecologic malignancies. This paper reviews the recent progression on Mettl3 in gynecologic malignancies, hoping to provide a reference for further research.

Keywords:  METTL3; N6‐methyladenosine; cancer therapy; gynecologic malignancies; m6A methyltransferase

DOI:  https://doi.org/10.3389/fphar.2023.1156629
Mol Med. 2023 Apr 03. 29(1): 46

m6A methyltransferase METTL3 programs CD4+ T-cell activation and effector T-cell differentiation in systemic lupus erythematosus.

Shuang Lu, Xingyu Wei, Huan Zhu, Zhi Hu, Meiling Zheng, Jiali Wu, Cheng Zhao, Shuang Yang, Delong Feng, Sujie Jia, Hongjun Zhao, Ming Zhao.

   BACKGROUND: Systemic lupus erythematosus (SLE) is an autoimmune disorder in which excessive CD4+ T-cell activation and imbalanced effector T-cell differentiation play critical roles. Recent studies have implied a potential association between posttranscriptional N6-methyladenosine (m6A) modification and CD4+ T-cell-mediated humoral immunity. However, how this biological process contributes to lupus is not well understood. In this work, we investigated the role of the m6A methyltransferase like 3 (METTL3) in CD4+ T-cell activation, differentiation, and SLE pathogenesis both in vitro and in vivo.
METHODS: The expression of METTL3 was knocked down and METTL3 enzyme activity was inhibited using siRNA and catalytic inhibitor, respectively. In vivo evaluation of METTL3 inhibition on CD4+ T-cell activation, effector T-cell differentiation, and SLE pathogenesis was achieved using a sheep red blood cell (SRBC)-immunized mouse model and a chronic graft versus host disease (cGVHD) mouse model. RNA-seq was performed to identify pathways and gene signatures targeted by METTL3. m6A RNA-immunoprecipitation qPCR was applied to confirm the m6A modification of METTL3 targets.
RESULTS: METTL3 was defective in the CD4+ T cells of SLE patients. METTL3 expression varied following CD4+ T-cell activation and effector T-cell differentiation in vitro. Pharmacological inhibition of METTL3 promoted the activation of CD4+ T cells and influenced the differentiation of effector T cells, predominantly Treg cells, in vivo. Moreover, METTL3 inhibition increased antibody production and aggravated the lupus-like phenotype in cGVHD mice. Further investigation revealed that catalytic inhibition of METTL3 reduced Foxp3 expression by enhancing Foxp3 mRNA decay in a m6A-dependent manner, hence suppressing Treg cell differentiation.
CONCLUSION: In summary, our findings demonstrated that METTL3 was required for stabilizing Foxp3 mRNA via m6A modification to maintain the Treg differentiation program. METTL3 inhibition contributed to the pathogenesis of SLE by participating in the activation of CD4+ T cells and imbalance of effector T-cell differentiation, which could serve as a potential target for therapeutic intervention in SLE.

Keywords:  Autoimmune disorders; METTL3; N6-Methyladenosine; SLE; mRNA methylation

DOI:  https://doi.org/10.1186/s10020-023-00643-4
Front Immunol. 2023 ;14 1128459

Comprehensive analysis of m6A RNA methylation modification patterns and the immune microenvironment in osteoarthritis.

Zhixin Liu, Heng Liu, Deqiang Li, Liang Ma, Tongxin Lu, Hao Sun, Yuankai Zhang, Hui Yang.

   Background: Osteoarthritis (OA) is the most common joint degenerative disease, and so far, there is no effective therapy to prevent or delay its development. Considerable attention is now being given to the impact of m6A RNA methylation modification on the disease immune regulation. However, much remains unknown about the function of m6A modification in OA.
Methods: A total of 63 OA and 59 healthy samples were applied to comprehensively examine the m6A regulators mediated RNA methylation modification pattern in OA, and evaluate the impacts of distinct patterns on the characteristics of OA immune microenvironment, including immune infiltration cells, immune responses and human leukocyte antigen (HLAs) genes expression. In addition, we screened out the m6A phenotype-related genes and further explored their potential biological functions. At last, we verified the expression of key m6A regulators and their associations with immune cells, in vitro.
Results: Most of m6A regulators was differentially expressed in OA samples compared to the normal tissues. Based on six hub-m6A regulators identified as abnormally expressed in OA samples, we developed a classifier to distinguish OA patients from healthy individuals. We noted that immune characteristics of OA were correlated with m6A regulators. For instance, YTHDF2 had a strongest significantly positive correlation with regulatory T cells (Tregs) and IGFBP2 was strongest negatively associated with dendritic cells (DCs), which were confirmed by the immunohistochemistry (IHC) staining. Two distinct m6A modification patterns were determined: pattern B had higher infiltrating immunocytes and more active immune responses than pattern A, and two patterns differed in the expression of HLA genes. We also identified 1,592 m6A phenotype-related genes that could mediate the OA synovitis and cartilage degradation by the PI3K-Akt signaling pathway. Quantitative real-time polymerase chain reaction (qRT-PCR) results indicated that IGFBP2 was significantly overexpressed, while YTHDF2 mRNA expression was decreased in OA samples, which was consistent with our findings.
Conclusion: Our research proves the essential impact of m6A RNA methylation modification on the OA immune microenvironment, and helps to explain the regulatory mechanism behind it, which may open up a new direction for more precise immunotherapy of osteoarthritis.

Keywords:  bioinformatics; epigenetics; immune microenvironment; m6A RNA methylation; osteoarthritis

DOI:  https://doi.org/10.3389/fimmu.2023.1128459
J Biol Chem. 2023 Apr 05. pii: S0021-9258(23)00319-8. [Epub ahead of print] 104677

VIRMA Promotes Nasopharyngeal Carcinoma Tumorigenesis and Metastasis by Upregulation of E2F7 in an m6A-Dependent Manner.

Zi-Qi Zheng, Zhuo-Hui Huang, Ye-Lin Liang, Wei-Hong Zheng, Cheng Xu, Zhi-Xuan Li, Na Liu, Pan-Yang Yang, Ying-Qin Li, Jun Ma, Ying Sun, Ling-Long Tang, Denghui Wei.

  The N6-methyladenosine (m6A) modification possesses new and essential roles in tumor initiation and progression by regulating mRNA biology. However, the role of aberrant m6A regulation in nasopharyngeal carcinoma (NPC) remains unclear. Here, through comprehensive analyses of NPC cohorts from the GEO database and our internal cohort, we identified that VIRMA, an m6A writer, is significantly upregulated in NPC and plays an essential role in tumorigenesis and metastasis of NPC, both in vitro and in vivo. High VIRMA expression served as a prognostic biomarker and was associated with poor outcomes in patients with NPC. Mechanistically, VIRMA mediated the m6A methylation of E2F7 3'-UTR, then IGF2BP2 bound and maintained the stability of E2F7 mRNA. An integrative high-throughput sequencing approach revealed that E2F7 drives a unique transcriptome distinct from the classical E2F family in NPC, which functioned as an oncogenic transcriptional activator. E2F7 cooperated with CBFB-recruited RUNX1 in a non-canonical manner to transactivate ITGA2, ITGA5, and NTRK1, strengthening Akt signaling-induced tumor-promoting effect.

Keywords:  E2F7; Nasopharyngeal carcinoma; VIRMA; m6A; metastasis

DOI:  https://doi.org/10.1016/j.jbc.2023.104677
Stem Cells Transl Med. 2023 Apr 03. pii: szad020. [Epub ahead of print]

WTAP-Mediated m6A RNA Methylation Regulates the Differentiation of Bone Marrow Mesenchymal Stem Cells via the miR-29b-3p/HDAC4 Axis.

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

  N6-methyladenosine (m6A) methylation, a well-known modification with new epigenetic functions, has been reported to participate in the progression of osteoporosis (OP), providing novel insights into the pathogenesis of OP. However, as the key component of m6A methylation, Wilms tumor 1-associated protein (WTAP) has not been studied in OP. Here we explored the biological role and underlying mechanism of WTAP in OP and the differentiation of bone marrow mesenchymal stem cells (BMMSCs). We demonstrated that WTAP was expressed at low levels in bone specimens from patients with OP and OVX mice. Functionally, WTAP promoted osteogenic differentiation and inhibited adipogenic differentiation of BMMSCs in vitro and in vivo. In addition, microRNA-29b-3p (miR-29b-3p) was identified as a downstream target of WTAP. M6A modifications regulated by WTAP led to increased miR-29b-3p expression. WTAP interacted with the microprocessor protein DGCR8 and accelerated the maturation of pri-miR-29b-3p in an m6A-dependent manner. Target prediction and dual-luciferase reporter assays identified the direct binding sites of miR-29b-3p with histone deacetylase 4 (HDAC4). WTAP-mediated m6A modification promoted osteogenic differentiation and inhibited adipogenic differentiation of BMMSCs through the miR-29b-3p/HDAC4 axis. Furthermore, WTAP-mediated m6A methylation negatively regulates osteoclast differentiation. Collectively, our study first identified a critical role of WTAP-mediated m6A methylation in BMMSC differentiation and highlighted WTAP as a potential therapeutic target for OP treatment.

Keywords:  BMMSCs; HDAC4; WTAP; adipogenesis; m6A; miR-29b-3p; osteogenesis

DOI:  https://doi.org/10.1093/stcltm/szad020
Oncogene. 2023 Apr 03.

N6-methyladenosine reader YTHDF2 promotes multiple myeloma cell proliferation through EGR1/p21cip1/waf1/CDK2-Cyclin E1 axis-mediated cell cycle transition.

Rui Liu, Jiyu Miao, Yachun Jia, Guangyao Kong, Fei Hong, Fangmei Li, Meng Zhai, Ru Zhang, Jiaxi Liu, Xuezhu Xu, Ting Wang, Hui Liu, Jinsong Hu, Yun Yang, Aili He.

Multiple myeloma (MM) is the second most common hematological malignancy. N6-methyladenosine (m6A) is the most abundant RNA modification. YTH domain-containing family protein 2 (YTHDF2) recognizes m6A-cotaining RNAs and accelerates degradation to regulate cancer progression. However, the role of YTHDF2 in MM remains unclear. We investigated the expression levels and prognostic role of YTHDF2 in MM, and studied the effect of YTHDF2 on MM proliferation and cell cycle. The results showed that YTHDF2 was highly expressed in MM and was an independent prognostic factor for MM survival. Silencing YTHDF2 suppressed cell proliferation and caused the G1/S phase cell cycle arrest. RNA immunoprecipitation (RIP) and m6A-RIP (MeRIP) revealed that YTHDF2 accelerated EGR1 mRNA degradation in an m6A-dependent manner. Moreover, overexpression of YTHDF2 promoted MM growth via the m6A-dependent degradation of EGR1 both in vitro and in vivo. Furthermore, EGR1 suppressed cell proliferation and retarded cell cycle by activating p21cip1/waf1 transcription and inhibiting CDK2-cyclinE1. EGR1 knockdown could reverse the inhibited proliferation and cell cycle arrest upon YTHDF2 knockdown. In conclusion, the high expression of YTHDF2 promoted MM cell proliferation via EGR1/p21cip1/waf1/CDK2-cyclin E1 axis-mediated cell cycle transition, highlighting the potential of YTHDF2 as an effective prognostic biomarker and a promising therapeutic target for MM.

DOI: https://doi.org/10.1038/s41388-023-02675-w
J Ethnopharmacol. 2023 Mar 31. pii: S0378-8741(23)00301-X. [Epub ahead of print] 116433

Chinese Ecliptae herba (Eclipta prostrata (L.) L.) extract and its component wedelolactone enhances osteoblastogenesis of bone marrow mesenchymal stem cells via targeting METTL3-mediated m6A RNA methylation.

Shuo Tian, Yi-Lin Li, Jie Wang, Ren-Chao Dong, Jun Wei, Yu Ma, Yan-Qiu Liu.

   ETHNOPHARMACOLOGICAL RELEVANCE: Chinese Ecliptae herba (Eclipta prostrata (L.) L.) is an ethnomedicinal herb, which is used mainly to nourish kidney and thus strengthen bones according to traditional Chinese medicine theory. Pharmacological studies have supported the ethnomedicine use, showing that Ecliptae herba extract has an anti-osteoporotic effect in vivo and promoted osteoblast proliferation and activity in vitro. However, the molecular mechanism of Ecliptae herba on osteoblast differentiation from bone marrow mesenchymal stem cells (BMSC), the progenitors of osteoblasts, is still unclear.
AIM OF THE STUDY: N6-methyladenosine (m6A) mRNA epigenetic modification may play a key role in promoting osteoblastic differentiation, and thus treating osteoporosis. This study sought to assess the mechanism through which Eclipate herba and its component wedelolactone influence m6A modification during the process of osteoblastogenesis from BMSC.
MATERIAL AND METHODS: The alkaline phosphatase (ALP) and Alizarin red S (ARS) staining were applied to determine osteoblastogenesis from BMSC. Western blot and quantitative real-time PCR were performed. RNA sequencing analysis was used to determine the characteristics of m6A methylation. Stable knocking down of METTL3 using lentiviral-based shRNA was performed.
RESULTS: Upon 9 d treatment of BMSC with ethyl acetate extract of Ecliptae herba (MHL), ALP activity and ossification level increased in comparison with osteogenic medium (OS)-treated control. The expression of methyltransferase METTL3 and METTL14 was significantly increased, but WTAP expression had no change in response to MHL treatment. Knocking down of METTL3 resulted in a decrease in MHL-induced ALP activity, ossification level as well as mRNA expression of Osterix and Osteocalcin, two bone formation-related markers. The level of m6A increased when BMSC was treated with MHL for 9 d. RNA sequencing analysis indicated that MHL treatment altered mRNA m6A modification of genes associated with osteoblastogenesis. By kyoto encyclopedia of genes and genomes (KEGG) pathway analysis, HIF-1α, PI3K/Akt, and Hippo signaling pathways were enriched and associated with m6A modification. The expression of m6A-modified genes including HIF-1α, VEGF-A, and RASSF1, was upregulated by MHL, but the upregulation was reversed after METTL3 knockdown. Additionally, the enhanced expression of METTL3 was also observed after treatment with wedelolactone, a component from MHL.
CONCLUSIONS: These results suggested a previously uncharacterized mechanism of MHL and wedelolactone on osteoblastogenesis, by which METTL3-mediated m6A methylation is involved and thus contributes to the enhancement of osteoblastogenesis.

Keywords:  BMSC; Ecliptae herba; METTL3; N6-methyladenosine; Osteoblastogenesis

DOI:  https://doi.org/10.1016/j.jep.2023.116433
Biochim Biophys Acta Mol Basis Dis. 2023 Apr 03. pii: S0925-4439(23)00074-1. [Epub ahead of print] 166708

WTAP promotes macrophage recruitment and increases VEGF secretion via N6-methyladenosine modification in corneal neovascularization.

Yanhui Bai, Xiaohang Jiao, Jinge Hu, Wenxin Xue, Ziyu Zhou, Weiqun Wang.

   BACKGROUND: Corneal neovascularization (CNV) can be caused by chemical burns. Macrophages are involved in angiogenesis and lymphangiogenesis during CNV. The aim of this study was to investigate whether Wilms' tumor 1-associated protein (WTAP) is involved in macrophage recruitment and VEGF secretion via N6-methyladenosine (m6A) modification.
METHODS: A CNV mouse model was established by corneal alkali burn. Tumor necrosis factor alpha (TNF-α) was used to stimulate vascular endothelial cells. m6A immunoprecipitation qPCR was used to determine the enrichment of m6A levels in mRNAs. The H3K9me3 enrichment in the promoter region of CC motif chemokine ligand 2 (CCL2) was detected by chromatin immunoprecipitation assay. The WTAP inhibition in vivo was performed using the adeno-associated virus.
RESULTS: In the alkali burn corneal tissues, angiogenesis and lymphangiogenesis were promoted as CD31 and LYVE-1 expressions were elevated, and the number of macrophages as well as WTAP expression were increased. Under the TNF-α stimulation, WTAP promoted the recruitment of endothelial cells to macrophages by promoting CCL2 secretion. Mechanistically, WTAP affected the enrichment of H3K9me3 at the CCL2 promoter by regulating the m6A level of SUV39H1 mRNA. The in vivo experiment showed that VEGFA/C/D secretion of macrophages was reduced after WTAP interference. Mechanistically, WTAP regulated the translational efficiency of HIF-1α via m6A modification.
CONCLUSION: WTAP affected macrophage recruitment to endothelial cells via regulation of H3K9me3-mediated CCL2 transcription. WTAP also affected macrophage secretion of VEGFA/C/D via m6A-mediated translation regulation of HIF-1α. Both pathways were involved in the WTAP regulation of angiogenesis and lymphangiogenesis during CNV.

Keywords:  Angiogenesis; Corneal neovascularization; Lymphangiogenesis; Macrophage; m6A

DOI:  https://doi.org/10.1016/j.bbadis.2023.166708
Clin Epigenetics. 2023 Apr 07. 15(1): 60

Comprehensive analyses of molecular features, prognostic values, and regulatory functionalities of m6A-modified long non-coding RNAs in lung adenocarcinoma.

Yili Ping, Jingjuan Huang, Jichao Zhu, Zujun Sun, Anquan Shang, Chen Chen, Wenfang Liu, Dong Li.

   BACKGROUND: Lung adenocarcinoma (LUAD) has a high incidence and recurrence rate. N6-methyladenosine (m6A) modification of RNA has become a promising epigenetic marker in tumors. The dysregulation of both RNA m6A levels and m6A regulator expression levels reportedly affects essential biological processes in various tumors. Long non-coding RNAs (lncRNAs), a subgroup of RNAs over 200 nucleotides in length that do not code for protein, can be modified and regulated by m6A, but the relevant profile in LUAD remains unclear.
RESULTS: The m6A levels of total RNA were decreased in LUAD tumor tissues and cells. Multiple m6A regulators were abnormally expressed at both the RNA and protein levels, and were related in expression patterns and functionally synergistic. Our microarray revealed 2846 m6A-modified lncRNA transcripts as well as its molecular features, 143 of which were differentially m6A-modified and manifested a negative correlation between expression levels and m6A modification levels. More than half of the differentially m6A-modified lncRNAs associated with dysregulated expression. The 6-MRlncRNA risk signature was a reliable indicator for assessing survival time of LUAD patients. The competitive endogenous regulatory network suggested a potential m6A-induced pathogenicity in LUAD.
CONCLUSIONS: These data have demonstrated that differential RNA m6A modification and m6A regulator expression levels were identified in LUAD patients. In addition, this study provides evidence increasing the understanding of molecular features, prognostic values, and regulatory functionalities of m6A-modified lncRNAs in LUAD.

Keywords:  Bioinformatics analysis; LncRNA; Lung adenocarcinoma; N6-methyladenosine; Prognostic values

DOI:  https://doi.org/10.1186/s13148-023-01475-z
Tissue Cell. 2023 Mar 26. pii: S0040-8166(23)00069-1. [Epub ahead of print]82 102081

Role of N6-adenosine-methyltransferase subunits METTL3 and METTL14 in the biological properties of periodontal ligament cells.

Jing Huang, Chen Guo, Yining Wang, Yi Zhou.

  The N6-methyladenosine (m6A) modification has been proven to be involved in various physiological and pathological processes. The m6A is catalyzed by methyltransferase complex, which mainly consist of methyltransferase (METTL) 3 and 14 heterodimer. The present study aimed to investigate the role of METTL 3 and 14 in biological properties of periodontal ligament cells (PDLCs) via RNA-sequencing and specific cell assays. Firstly, the expressions of METTL3 and METTL14 were observed in PDLCs. Then, RNA-sequencing showed that cell properties were influenced after METTL3 or METTL14 was knocked down via short hairpin RNA (shRNA). In sh-METTL3 or METTL14 PDLCs, cell counting kit 8 (CCK8) and 5-ethynyl-2'-deoxyuridine (EdU) assays showed a down-regulated proliferation, transwell system indicated suppressed migration. Lastly, alkaline phosphatase (ALP) and alizarin red staining (ARS) staining, quantitative polymerase chain reaction (qPCR) and western blot demonstrated the inhibited osteogenic potentials. It could be concluded that METTL3 and METTL14 play indispensable roles in the regenerative potential of PDLCs.

Keywords:  Epigenetics; M6A; Periodontium; Regenerative potential; Short hairpin RNA

DOI:  https://doi.org/10.1016/j.tice.2023.102081
J Anim Sci Biotechnol. 2023 Apr 06. 14(1): 50

Profiling of N6-methyladenosine methylation in porcine longissimus dorsi muscle and unravelling the hub gene ADIPOQ promotes adipogenesis in an m6A-YTHDF1-dependent manner.

Huanfa Gong, Tao Gong, Youhua Liu, Yizhen Wang, Xinxia Wang.

   BACKGROUND: Intramuscular fat (IMF) content is a critical indicator of pork quality, and abnormal IMF is also relevant to human disease as well as aging. Although N6-methyladenosine (m6A) RNA modification was recently found to regulate adipogenesis in porcine intramuscular fat, however, the underlying molecular mechanisms was still unclear.
RESULTS: In this work, we collected 20 longissimus dorsi muscle samples with high (average 3.95%) or low IMF content (average 1.22%) from a unique heterogenous swine population for m6A sequencing (m6A-seq). We discovered 70 genes show both differential RNA expression and m6A modification from high and low IMF group, including ADIPOQ and SFRP1, two hub genes inferred through gene co-expression analysis. Particularly, we observed ADIPOQ, which contains three m6A modification sites within 3' untranslated and protein coding region, could promote porcine intramuscular preadipocyte differentiation in an m6A-dependent manner. Furthermore, we found the YT521‑B homology domain family protein 1 (YTHDF1) could target and promote ADIPOQ mRNA translation.
CONCLUSIONS: Our study provided a comprehensive profiling of m6A methylation in porcine longissimus dorsi muscle and characterized the involvement of m6A epigenetic modification in the regulation of ADIPOQ mRNA on IMF deposition through an m6A-YTHDF1-dependent manner.

Keywords:  ADIPOQ; Intramuscular fat; N6-methyladenosine; Pig; YTHDF1

DOI:  https://doi.org/10.1186/s40104-023-00833-4
Cell Death Dis. 2023 Apr 04. 14(4): 236

Aberrant RNA m6A modification in gastrointestinal malignancies: versatile regulators of cancer hallmarks and novel therapeutic opportunities.

Li-Ting Shen, Lin-Rong Che, Zongsheng He, Qian Lu, Dong-Feng Chen, Zhong-Yi Qin, Bin Wang.

Gastrointestinal (GI) cancer is one of the most common malignancies, and a leading cause of cancer-related death worldwide. However, molecular targeted therapies are still lacking, leading to poor treatment efficacies. As an important layer of epigenetic regulation, RNA N6-Methyladenosine (m6A) modification is recently linked to various biological hallmarks of cancer by orchestrating RNA metabolism, including RNA splicing, export, translation, and decay, which is partially involved in a novel biological process termed phase separation. Through these regulatory mechanisms, m6A dictates gene expression in a dynamic and reversible manner and may play oncogenic, tumor suppressive or context-dependent roles in GI tumorigenesis. Therefore, regulators and effectors of m6A, as well as their modified substrates, represent a novel class of molecular targets for cancer treatments. In this review, we comprehensively summarize recent advances in this field and highlight research findings that documented key roles of RNA m6A modification in governing hallmarks of GI cancers. From a historical perspective, milestone findings in m6A machinery are integrated with a timeline of developing m6A targeting compounds. These available chemical compounds, as well as other approaches that target core components of the RNA m6A pathway hold promises for clinical translational to treat human GI cancers. Further investigation on several outstanding issues, e.g. how oncogenic insults may disrupt m6A homeostasis, and how m6A modification impacts on the tumor microenvironment, may dissect novel mechanisms underlying human tumorigenesis and identifies next-generation anti-cancer therapeutics. In this review, we discuss advances in our understanding of m6A RNA modification since its discovery in the 1970s to the latest progress in defining its potential clinic relevance. We summarize the molecular basis and roles of m6A regulators in the hallmarks of GI cancer and discuss their context-dependent functions. Furthermore, the identification and characterization of inhibitors or activators of m6A regulators and their potential anti-cancer effects are discussed. With the rapid growth in this field there is significant potential for developing m6A targeted therapy in GI cancers.

DOI: https://doi.org/10.1038/s41419-023-05736-w
Nat Commun. 2023 Apr 05. 14(1): 1898

The m6A reader YTHDC1 and the RNA helicase DDX5 control the production of rhabdomyosarcoma-enriched circRNAs.

Dario Dattilo, Gaia Di Timoteo, Adriano Setti, Andrea Giuliani, Giovanna Peruzzi, Manuel Beltran Nebot, Alvaro Centrón-Broco, Davide Mariani, Chiara Mozzetta, Irene Bozzoni.

N6-Methyladenosine (m6A) is well-known for controlling different processes of linear RNA metabolism. Conversely, its role in the biogenesis and function of circular RNAs (circRNAs) is still poorly understood. Here, we characterize circRNA expression in the pathological context of rhabdomyosarcoma (RMS), observing a global increase when compared to wild-type myoblasts. For a set of circRNAs, such an increase is due to the raised expression of the m6A machinery, which we also find to control the proliferation activity of RMS cells. Furthermore, we identify the RNA helicase DDX5 as a mediator of the back-splicing reaction and as a co-factor of the m6A regulatory network. DDX5 and the m6A reader YTHDC1 are shown to interact and to promote the production of a common subset of circRNAs in RMS. In line with the observation that YTHDC1/DDX5 depletion reduces RMS proliferation, our results provide proteins and RNA candidates for the study of rhabdomyosarcoma tumorigenicity.

DOI: https://doi.org/10.1038/s41467-023-37578-7
Genome Biol. 2023 Apr 06. 24(1): 67

Reading and writing of mRNA m6A modification orchestrate maternal-to-zygotic transition in mice.

Wencheng Zhu, Yufeng Ding, Juan Meng, Lei Gu, Wenjun Liu, Li Li, Hongyu Chen, Yining Wang, Ziyi Li, Chen Li, Yidi Sun, Zhen Liu.

  N6-methyladenosine (m6A) modification has been shown to regulate RNA metabolism. Here, we investigate m6A dynamics during maternal-to-zygotic transition (MZT) in mice through multi-omic analysis. Our results show that m6A can be maternally inherited or de novo gained after fertilization. Interestingly, m6A modification on maternal mRNAs not only correlates with mRNA degradation, but also maintains the stability of a small group of mRNAs thereby promoting their translation after fertilization. We identify Ythdc1 and Ythdf2 as key m6A readers for mouse preimplantation development. Our study reveals a key role of m6A mediated RNA metabolism during MZT in mammals.

Keywords:  Maternal-to-zygotic transition; Multi-omics; Ythdc1; Ythdf2; m6A landscape

DOI:  https://doi.org/10.1186/s13059-023-02918-9
Front Endocrinol (Lausanne). 2023 ;14 1155009

Single-cell mapping of N6-methyladenosine in esophageal squamous cell carcinoma and exploration of the risk model for immune infiltration.

Yuanliu Nie, Guangyue Yao, Xiaoying Xu, Yi Liu, Ke Yin, Jingjiang Lai, Qiang Li, Fengge Zhou, Zhe Yang.

   Background: N6-methyladenosine (m6A) modification is the most common RNA modification, but its potential role in the development of esophageal cancer and its specific mechanisms still need to be further investigated.
Methods: Bulk RNA-seq of 174 patients with esophageal squamous carcinoma from the TCGA-ESCC cohort, GSE53625, and single-cell sequencing data from patients with esophageal squamous carcinoma from GSE188900 were included in this study. Single-cell analysis of scRNA-seq data from GSE188900 of 4 esophageal squamous carcinoma samples and calculation of PROGENy scores. Demonstrate the scoring of tumor-associated pathways for different cell populations. Cell Chat was calculated for cell populations. thereafter, m6A-related differential genes were sought and risk models were constructed to analyze the relevant biological functions and impact pathways of potential m6A genes and their impact on immune infiltration and tumor treatment sensitivity in ESCC was investigated.
Results: By umap downscaling analysis, ESCC single-cell data were labelled into clusters of seven immune cell classes. Cellchat analysis showed that the network interactions of four signaling pathways, MIF, AFF, FN1 and CD99, all showed different cell type interactions. The prognostic risk model constructed by screening for m6A-related differential genes was of significant value in the prognostic stratification of ESCC patients and had a significant impact on immune infiltration and chemotherapy sensitivity in ESCC patients.
Conclusion: In our study, we explored a blueprint for the distribution of single cells in ESCC based on m6A methylation and constructed a risk model for immune infiltration analysis and tumor efficacy stratification in ESCC on this basis. This may provide important potential guidance for revealing the role of m6A in immune escape and treatment resistance in esophageal cancer.

Keywords:  N6-Methyladenosine; bioinfomatics; esophageal squamous cell carcinoma; immune infiltration; single-gene sequencing

DOI:  https://doi.org/10.3389/fendo.2023.1155009
Eur Rev Med Pharmacol Sci. 2023 Mar;pii: 31793. [Epub ahead of print]27(6): 2561-2570

METTL3-mediated downregulation of splicing factor SRSF11 is associated with carcinogenesis and poor survival of cancer patients.

J Oh, J-M Oh, S-Y Cho.

OBJECTIVE: N6-methyladenosine (m6A) is one of the most abundant post-transcriptional modifications in eukaryotic RNA. As m6A modifications play an important role in RNA processing, abnormal m6A regulation caused by aberrant expression of m6A regulators is closely related to carcinogenesis. In this study, we aimed to determine the role of METTL3 expression in carcinogenesis, regulation of splicing factor expression by METTL3, and their effects in survival period and cancer-related metabolisms.
MATERIALS AND METHODS: We investigated the correlation between each splicing factor and METTL3 in breast invasive ductal carcinoma (BRCA), colon adenocarcinoma (COAD), lung adenocarcinoma (LUAD) and gastric adenocarcinoma (STAD). Survival analysis was performed based on the expression of each splicing factor. To determine the molecular mechanism of SRSF11 in carcinogenesis, gene set enrichment analysis using RNA sequencing data was performed according to SRSF11 expression.
RESULTS: Among the 64 splicing factors used for correlation analysis, 13 splicing factors showed a positive correlation with METTL3 in all four cancer types. We found that when METTL3 expression was decreased, the expression of SRSF11 was also decreased in all four types of cancer tissue when compared to that in normal tissue. Decreased SRSF11 expression was associated with poor survival in patients with BRCA, COAD, LUAD, and STAD. Gene set enrichment analysis according to SRSF11 expression showed that the p53/apoptosis, inflammation/immune response, and ultraviolet/reactive oxygen species stimulus-response pathways were enriched in cancers with decreased SRSF11 expression.
CONCLUSIONS: These results suggest that METTL3 regulates SRSF11 expression, which could influence mRNA splicing in m6A modified cancer cells. METTL3-mediated downregulation of SRSF11 expression in cancer patients correlates with poor prognosis.

DOI: https://doi.org/10.26355/eurrev_202303_31793
Cancer Lett. 2023 Apr 05. pii: S0304-3835(23)00116-7. [Epub ahead of print] 216165

When N7-methyladenosine modification meets cancer: Emerging frontiers and promising therapeutic opportunities.

Dan Du, Jing He, Chenxi Ju, Chang Wang, Hongle Li, Fucheng He, Mingxia Zhou.

  N7-methylguanosine (m7G) methylation, one of the most common RNA modifications in eukaryotes, has recently gained considerable attention. The biological functions of m7G modification in RNAs, including tRNA, rRNA, mRNA, and miRNA, remain largely unknown in human diseases. Owing to rapid advances in high-throughput technologies, increasing evidence suggests that m7G modification plays a critical role in cancer initiation and progression. As m7G modification and hallmarks of cancer are inextricably linked together, targeting m7G regulators may provide new possibilities for future cancer diagnoses and potential intervention targets. This review summarizes various detection methods for m7G modification, recent advances in m7G modification and tumor biology regarding their interplay and regulatory mechanisms. We conclude with an outlook on the future of diagnosing and treating m7G-related diseases.

Keywords:  Cancer progression; METTL1; N(7)-methylguanosine (m(7)G); RNA modification; WDR4

DOI:  https://doi.org/10.1016/j.canlet.2023.216165
Aging (Albany NY). 2023 Mar 17. 15(6): 1944-1963

Development a m6A regulators characterized by the immune cell infiltration in stomach adenocarcinoma for predicting the prognosis and immunotherapy response.

Guodong Shi, Yang Li, Huijiang Gao, Yucheng Wei, Yuanyong Wang.

  N6-Methyladenosine (m6A) has attracted growing interest among scholars as an important regulator of mRNA expression. Although the significant role of m6A in multiple biological processes (like proliferation and growth of cancers) has been comprehensively described, an analysis of its possible role in stomach adenocarcinoma (STAD) of tumor immune microenvironment (TIME) remains lacking. The data for RNA expression, single nucleotide polymorphism (SNP), and copy number variation (CNV) were downloaded from The Cancer Genome Atlas (TCGA). Subsequently, 23 m6A regulators were curated, with patients being clustered into three m6A subtypes and m6A-related gene subtypes. Furthermore, they were compared based on overall survival (OS). This study also evaluates the association between m6A regulators and immune as well as response to the treatment. According to the TCGA-STAD cohort, three m6A clusters conformed to three phenotypes, immune-inflamed, immune-dessert, and immune-excluded, respectively. Patients who displayed lower m6A scores presented better overall survival outcomes. The GEO cohort demonstrated that those with a low m6A score had obvious general survival benefits and clinical advantages. Low m6A scores can carry the enhanced neoantigen loads, triggering an immune response. Meanwhile, three anti-PD-1 cohorts have confirmed the value of predicting survival outcomes. The results of this study indicate that m6A regulators are associated with TIME, and the m6A score is an efficient prognostic biomarker and predictive indicator for immunotherapy and chemotherapeutics. Moreover, comprehensive evaluations of m6A regulators in tumors will broaden our comprehension of TIME, efficiently guiding enhancing explorations on immunotherapy and chemotherapy strategies for STAD.

Keywords:  STAD; TIME; m6A regulator; m6A score; therapy

DOI:  https://doi.org/10.18632/aging.204574
Front Immunol. 2023 ;14 1019445

Expression patterns of eight RNA-modified regulators correlating with immune infiltrates during the progression of osteoarthritis.

Ziyi Chen, Wenjuan Wang, Yinghui Hua.

   Background: RNA modifications in eukaryotic cells have emerged as an exciting but under-explored area in recent years and are considered to be associated with many human diseases. While several studies have been published relating to m6A in osteoarthritis (OA), we only have limited knowledge of other kinds of RNA modifications. Our study investigated eight RNA modifiers' specific roles in OA including A-to-I, APA, m5C, m6A, m7G, mcm5s2U, Nm and Ψ together with their relationship with immune infiltration.
Methods: RNA modification patterns in OA samples were identified based on eight-type RNA modifiers and their correlation with the degree of immune infiltration was also methodically investigated. Receiver operating characteristic curves (ROC) and qRT-PCR was performed to confirm the abnormal expression of hub genes. The RNA modification score (Rmscore) was generated by the applications of principal component analysis (PCA) algorithm in order to quantify RNA modification modes in individual OA patients.
Results: We identified 21 differentially-expressed RNA modification related genes between OA and healthy samples. For example, CFI, CBLL1 and ALKBH8 were expressed at high levels in OA (P<0.001), while RPUSD4, PUS1, NUDT21, FBL and WDR4 were expressed at low levels (P<0.001). Two candidate RNA modification regulators (WDR4 and CFI) were screened out utilizing a random forest machine learning model. We then identified two distinctive RNA modification modes in OA which were found to display distinctive biological features. High Rmscore, characterized by increased immune cell infiltration, indicated an inflamed phenotype.
Conclusions: Our study was the first to systematically reveal the crosstalk and dysregulations eight-type of RNA modifications in OA. Assessing individuals' RNA modification patterns will be conductive to enhance our understanding of the properties of immune infiltration, provide novel diagnostic and prognostic biomarkers, and guide more effective immunotherapy strategies in the future.

Keywords:  RNA modification regulators; alternative polyadenylation (APA); immune infiltration; m6A methylation; m7G methylation; osteoarthritis (OA)

DOI:  https://doi.org/10.3389/fimmu.2023.1019445
Mutat Res Genet Toxicol Environ Mutagen. 2023 Apr;pii: S1383-5718(23)00026-8. [Epub ahead of print]887 503608

FTO regulates the DNA damage response via effects on cell-cycle progression.

Weiying Liu, Manabu Yasui, Akira Sassa, Xinyue You, Jingjing Wan, Yiyi Cao, Jing Xi, Xinyu Zhang, Masamitsu Honma, Yang Luan.

  The fat mass and obesity-associated protein FTO is an "eraser" of N6-methyladenosine, the most abundant mRNA modification. FTO plays important roles in tumorigenesis. However, its activities have not been fully elucidated and its possible involvement in DNA damage - the early driving event in tumorigenesis - remains poorly characterized. Here, we have investigated the role of FTO in the DNA damage response (DDR) and its underlying mechanisms. We demonstrate that FTO responds to various DNA damage stimuli. FTO is overexpressed in mice following exposure to the promutagens aristolochic acid I and benzo[a]pyrene. Knockout of the FTO gene in TK6 cells, via CRISPR/Cas9, increased genotoxicity induced by DNA damage stimuli (micronucleus and TK mutation assays). Cisplatin- and diepoxybutane-induced micronucleus frequencies and methyl methanesulfonate- and azathioprine-induced TK mutant frequencies were also higher in FTO KO cells. We investigated the potential roles of FTO in DDR. RNA sequencing and enrichment analysis revealed that FTO deletion disrupted the p38 MAPK pathway and inhibited the activation of nucleotide excision repair and cell-cycle-related pathways following cisplatin (DNA intrastrand cross-links) treatment. These effects were confirmed by western blotting and qRT-PCR. FTO deletion impaired cell-cycle arrest at the G2/M phase following cisplatin and diepoxybutane treatment (flow cytometry analysis). Our findings demonstrated that FTO is involved in several aspects of DDR, acting, at least in part, by impairing cell cycle progression.

Keywords:  Nucleotide excision repair; P38 MAPK

DOI:  https://doi.org/10.1016/j.mrgentox.2023.503608
J Environ Pathol Toxicol Oncol. 2023 ;42(3): 71-81

METTL14 Promotes Oral Squamous Cell Carcinoma Progression by Regulating the mRNA and m6A Levels of CALD1.

Ruixue Chen, Suxin Zhang, Hexiang Li, Mengyuan Yang, Yiwen Lu, Xudong Zhang.

Oral squamous cell carcinoma (OSCC) still threatens people's daily life. METTL14 is a newly discovered methyltransferase that catalyzes m6A methylation. Hence, this research was carried out to investigate the action mechanism of METTL14 in OSCC. The SCC-4 and UM2 cells, and tumorigenicity assay were utilized to investigate METTL14 roles in vitro and in vivo. Bioinformatic analysis was carried out with the UCSC, TCGA database and The Human Protein Atlas. The gene expression at mRNA and protein levels were measured by qRT-PCR and Western blot. In addition, cell growth and metastasis was analyzed by colony formation and transwell assays. MeRIP assay was performed to test the m6A levels of CALD1. The METTL14 and CALD1 levels were prominently expressed in OSCC cells. METTL14 silencing depleted the cell growth and metastasis. Furthermore, METTL14 silencing depleted the tumor growth in vivo. Additionally, the mRNA and m6A levels of CALD1 were depleted after METTL14 silencing. Overexpressed CALD1 neutralized the si-METTL14 effects in OSCC cells. In conclusion, METTL14 participated in the OSCC progression through modulating the mRNA and m6A levels of CALD1.

DOI: https://doi.org/10.1615/JEnvironPatholToxicolOncol.2022045134
Front Cell Dev Biol. 2023 ;11 1164706

N6-methyladenosine modifications in maternal-fetal crosstalk and gestational diseases.

Suqi Wu, Ketong Liu, Bingyan Zhou, Suwen Wu.

  As a medium among pregnant women, environment and fetus, placenta owns powerful and delicate epigenetic processes to regulate gene expression and maintain cellular homeostasis. N6-methyladenosine (m6A) is the most prevalent modification that determines the fate of RNA, and its dynamic reversibility indicates that m6A may serve as a sensitive responder to environmental stimuli. Emerging evidence suggests that m6A modifications play an essential role in placental development and maternal-fetal crosstalk, and are closely related to gestational diseases. Herein, we summarized the latest techniques for m6A sequencing and highlighted current advances of m6A modifications in maternal-fetal crosstalk and the underlying mechanisms in gestational diseases. Therefore, proper m6A modifications are important in placental development, but its disturbance mainly caused by various environmental factors can lead to abnormal placentation and function with possible consequences of gestational diseases, fetal growth and disease susceptibility in adulthood.

Keywords:  N6-methyladenosine; epigenetics; fetal growth; gestational diseases; maternal-fetal crosstalk

DOI:  https://doi.org/10.3389/fcell.2023.1164706
Nat Commun. 2023 Apr 06. 14(1): 1932

PRMT3-mediated arginine methylation of IGF2BP1 promotes oxaliplatin resistance in liver cancer.

Yunxing Shi, Yi Niu, Yichuan Yuan, Kai Li, Chengrui Zhong, Zhiyu Qiu, Keren Li, Zhu Lin, Zhiwen Yang, Dinglan Zuo, Jiliang Qiu, Wei He, Chenwei Wang, Yadi Liao, Guocan Wang, Yunfei Yuan, Binkui Li.

Although oxaliplatin-based chemotherapy has been effective in the treatment of hepatocellular carcinoma (HCC), primary or acquired resistance to oxaliplatin remains a major challenge in the clinic. Through functional screening using CRISPR/Cas9 activation library, transcriptomic profiling of clinical samples, and functional validation in vitro and in vivo, we identify PRMT3 as a key driver of oxaliplatin resistance. Mechanistically, PRMT3-mediated oxaliplatin-resistance is in part dependent on the methylation of IGF2BP1 at R452, which is critical for the function of IGF2BP1 in stabilizing the mRNA of HEG1, an effector of PRMT3-IGF2BP1 axis. Also, PRMT3 overexpression may serve as a biomarker for oxaliplatin resistance in HCC patients. Collectively, our study defines the PRTM3-IGF2BP1-HEG1 axis as important regulators and therapeutic targets in oxaliplatin-resistance and suggests the potential to use PRMT3 expression level in pretreatment biopsy as a biomarker for oxaliplatin-resistance in HCC patients.

DOI: https://doi.org/10.1038/s41467-023-37542-5
Front Genet. 2023 ;14 1127301

Analysis of eight types of RNA modification regulators and their correlation with the prognosis in hepatocellular carcinoma.

Wan Qin, Chen Jin, Jun Zou.

  RNA modification plays important role in the occurrence and development of hepatocellular carcinoma. The best characterized RNA modification is m6A, while other kinds of RNA modifications have not been fully investigated in hepatocellular carcinoma (HCC). In the current study, we investigated the roles of one hundred RNA modification regulators belonging to eight different types of cancer-related RNA modifications in HCC. Expression analysis revealed that nearly 90% RNA regulators exhibited significantly higher expression in tumors than normal tissues. By consensus clustering, we identified two clusters with distinct biological characteristics, immune microenvironment, and prognostic pattern. An RNA modification score (RMScore) was constructed and stratified patients into high- and low-risk group, which showed significantly different prognosis. Moreover, a nomogram including clinicopathologic features and the RMScore could well predict the survival in HCC patients. This study indicated the important role of eight types of RNA modification in HCC and develop a RMScore, which will be a new method to forecast the prognosis of HCC patients.

Keywords:  RNA modification; hepatocellular carcinoma; nomogram; prognosis; therapeutic responses

DOI:  https://doi.org/10.3389/fgene.2023.1127301
Mech Ageing Dev. 2023 Apr 04. pii: S0047-6374(23)00033-7. [Epub ahead of print] 111807

Contribution of A-to-I RNA editing, M6A RNA Methylation, and Alternative Splicing to physiological brain aging and neurodegenerative diseases.

Valentina Tassinari, Piergiorgio La Rosa, Eugenia Guida, Ambra Colopi, Sara Caratelli, Francesca De Paolis, Angela Gallo, Carlo Cenciarelli, Giuseppe Sconocchia, Susanna Dolci, Valeriana Cesarini.

  Aging is a physiological and progressive phenomenon in all organisms' life cycle, characterized by the accumulation of degenerative processes triggered by several alterations within molecular pathways. These changes compromise cell fate, resulting in the loss of functions in tissues throughout the body, including the brain. Physiological brain aging has been linked to structural and functional alterations, as well as to an increased risk of neurodegenerative diseases. Post-transcriptional RNA modifications modulate mRNA coding properties, stability, translatability, expanding the coding capacity of the genome, and are involved in all cellular processes. Among mRNA post-transcriptional modifications, the A-to-I RNA editing, m6A RNA Methylation and Alternative Splicing play a critical role in all the phases of a neuronal cell life cycle and alterations in their mechanisms of action significantly contribute to aging and neurodegeneration. Here we review our current understanding of the contribution of A-to-I RNA editing, m6A RNA Methylation, and Alternative Splicing to physiological brain aging process and neurodegenerative diseases.

Keywords:  A-to-I RNA editing; Alternative Splicing; brain aging; m6A RNA Methylation; neurodegenerative diseases

DOI:  https://doi.org/10.1016/j.mad.2023.111807
Life Sci. 2023 Apr 01. pii: S0024-3205(23)00293-X. [Epub ahead of print] 121659

Cuproptosis illustrates tumor micro-environment features and predicts prostate cancer therapeutic sensitivity and prognosis.

Bisheng Cheng, Chen Tang, Junjia Xie, Qianghua Zhou, Tianlong Luo, Qiong Wang, Hai Huang.

   BACKGROUND: Prostate cancer (PCA) is a common malignant genitourinary tumor that significantly impacts patient survival. Cuproptosis, a copper-dependent programmed cell death mechanism, plays a vital role in tumor development, therapy resistance, and immune microenvironment regulation in PCA. However, research on cuproptosis in prostate cancer is still in its early stages.
METHODS: Using the publicly available datasets TCGA and GEO, we first acquired the transcriptome and clinical information of PCA patients. The expression of cuprotosis-related genes (CRG) was identified and a prediction model was established based on LASSO-COX method. The predictive performance of this model was evaluated based on Kaplan-Meier method. Using GEO datasets, we further confirmed the critical genes level in the model. Tumor responses to immune checkpoint (ICP) inhibitors were predicted based on Tumor Immune Dysfunction and Exclusion (TIDE) score. The Genomics of Drug Sensitivity in Cancer (GDSC) was utilized to forecast drug sensitivity in cancer cells, whereas the GSVA was employed to analyze enriched pathways related to the cuproptosis signature. Subsequently, the function of PDHA1 gene in PCA was verified.
RESULTS: A predictive risk model on basis of five cuproptosis-related genes (ATP7B, DBT, LIPT1, GCSH, PDHA1) were established. The progression free survival of low-risk group was obviously longer than the high-risk group, and exhibit better response to ICB therapy.Furthermore,PDHA1 is very important in the pathological process of PCA according to regressions analysis result, and the validation of external data sets were conducted. High PDHA1 expression patients with PCA not only had a shorter PFS and were less likely to benefit from ICB treatment, but they were also less responsive to multiple targeted therapeutic drugs. In preliminary research, PDHA1 knockdown significantly decreased the proliferation and invasion of PCA cells.
CONCLUSION: This study established a novel cuproptosis-related gene-based prostate cancer prediction model that accurately predicts the prognosis of PCA patients. The model benefits individualized therapy and can assist clinicians in making clinical decisions for PCA patients. Furthermore, our data show that PDHA1 promotes PCA cell proliferation and invasion while modulating the susceptibility to immunotherapy and other targeted therapies. PDHA1 can be regarded as an important target for PCA therapy. This study conforms to the standards of cancer research and is linguistically fluent and meets native language standards.

Keywords:  Cuproptosis; Immunotherapy; PDHA1; Prognosis; Prostate cancer

DOI:  https://doi.org/10.1016/j.lfs.2023.121659
Nat Commun. 2023 Apr 05. 14(1): 1906

Systematic comparison of tools used for m6A mapping from nanopore direct RNA sequencing.

Zhen-Dong Zhong, Ying-Yuan Xie, Hong-Xuan Chen, Ye-Lin Lan, Xue-Hong Liu, Jing-Yun Ji, Fu Wu, Lingmei Jin, Jiekai Chen, Daniel W Mak, Zhang Zhang, Guan-Zheng Luo.

N6-methyladenosine (m6A) has been increasingly recognized as a new and important regulator of gene expression. To date, transcriptome-wide m6A detection primarily relies on well-established methods using next-generation sequencing (NGS) platform. However, direct RNA sequencing (DRS) using the Oxford Nanopore Technologies (ONT) platform has recently emerged as a promising alternative method to study m6A. While multiple computational tools are being developed to facilitate the direct detection of nucleotide modifications, little is known about the capabilities and limitations of these tools. Here, we systematically compare ten tools used for mapping m6A from ONT DRS data. We find that most tools present a trade-off between precision and recall, and integrating results from multiple tools greatly improve performance. Using a negative control could improve precision by subtracting certain intrinsic bias. We also observed variation in detection capabilities and quantitative information among motifs, and identified sequencing depth and m6A stoichiometry as potential factors affecting performance. Our study provides insight into the computational tools currently used for mapping m6A based on ONT DRS data and highlights the potential for further improving these tools, which may serve as the basis for future research.

DOI: https://doi.org/10.1038/s41467-023-37596-5
Oncogene. 2023 Apr 07.

Phase separation of DDX21 promotes colorectal cancer metastasis via MCM5-dependent EMT pathway.

Huabin Gao, Huiting Wei, Yang Yang, Hui Li, Jiangtao Liang, Jiecheng Ye, Fenfen Zhang, Liyuan Wang, Huijuan Shi, Jia Wang, Anjia Han.

RNA binding proteins (RBPs) contributes to cancer progression, but the underlying mechanism reminds unclear. Here, we find that DDX21, a representative RBP, is highly expressed in colorectal cancer (CRC), which leads to CRC cell migration and invasion in vitro, and CRC to liver metastasis and lung metastasis in vivo. This effect of DDX21 on CRC metastasis is correlated to the activation of Epithelial-mesenchymal transition (EMT) pathway. Moreover, we reveal that DDX21 protein is phase separated in vitro and in CRC cells, which controls CRC metastasis. Phase-separated DDX21 highly binds on MCM5 gene locus, which is markedly reduced when phase separation is disrupted by mutations on its intrinsically disordered region (IDR). The impaired metastatic ability of CRC upon DDX21 loss is restored by ectopic expression of MCM5, indicating MCM5 is a key downstream target of DDX21 for CRC metastasis. Furthermore, co-higher expressions of DDX21 and MCM5 is significantly correlated with poor survival outcomes of stage III and IV CRC patients, indicating the importance of this mechanism in CRC late and metastatic stage. Altogether, our results elucidate a new model of DDX21 in regulating CRC metastasis via phase separation.

DOI: https://doi.org/10.1038/s41388-023-02687-6
Pharmgenomics Pers Med. 2023 ;16 253-261

RUNX2 Reverses p53-Induced Chemotherapy Resistance in Gastric Cancer.

Yuan Huang, Lu Liang, Yong-Xiang Zhao, Bi-Hui Yao, Rui-Min Zhang, Lei Song, Zhong-Tao Zhang.

   Objective: Gastric cancer is one of the most common malignancies worldwide; however, its overall mortality has not improved significantly over the last decade. Chemoresistance plays a critical role in this issue. This study aimed to clarify the role and mechanism of runt-related transcription factor 2 (RUNX2) in platinum-based chemotherapy resistance.
Methods: First, a drug-resistant model of gastric cancer cells was established to evaluate the relative expression level of the RUNX2 as a potential biomarker of chemotherapy resistance. Next, exogenous silencing was conducted to study whether RUNX2 could reverse drug resistance and understand the underlying mechanisms. Simultaneously, the correlation between the clinical outcomes of 40 patients after chemotherapy and the RUNX2 expression levels in tumor samples was analyzed.
Results: We discovered that RUNX2 was significantly expressed in drug-resistant gastric cancer cells and tissues; it was also reversibly resistant to transformation treatment by exogenous RUNX2 silencing. It is confirmed that RUNX2 negatively regulates the apoptosis pathway of the p53 to reduce the chemotherapeutic effects of gastric cancer.
Conclusion: RUNX2 is a possible target for platinum-based chemotherapy resistance.

Keywords:  RUNX2; chemotherapy resistance; gastric cancer; platinum

DOI:  https://doi.org/10.2147/PGPM.S394393
Front Immunol. 2023 ;14 1076587

Characterization of heterogeneous metabolism in hepatocellular carcinoma identifies new therapeutic target and treatment strategy.

Jiabin Yang, Liangtang Zeng, Ruiwan Chen, Shangyou Zheng, Yu Zhou, Rufu Chen.

   Background: Metabolic reprogramming is a well-known hallmark of cancer. Systematical identification of clinically relevant metabolic subtypes of Hepatocellular carcinoma (HCC) is critical to understand tumor heterogeneity and develop efficient treatment strategies.
Methods: We performed an integrative analysis of genomic, transcriptomic, and clinical data from an HCC patient cohort in The Cancer Genome Atlas (TCGA).
Results: Four metabolic subtypes were defined: mHCC1, mHHC2, mHCC3, and mHCC4. These subtypes had distinct differences in mutations profiles, activities of metabolic pathways, prognostic metabolism genes, and immune features. The mHCC1 was associated with poorest outcome and was characterized by extensive metabolic alterations, abundant immune infiltration, and increased expression of immunosuppressive checkpoints. The mHHC2 displayed lowest metabolic alteration level and was associated with most significant improvement in overall survival in response to high CD8+ T cell infiltration. The mHHC3 was a "cold-tumor" with low immune infiltration and few metabolic alterations. The mHCC4 presented a medium degree of metabolic alteration and high CTNNB1 mutation rate. Based on our HCC classification and in vitro study, we identified palmitoyl-protein thioesterase 1 (PPT1) was a specific prognostic gene and therapeutic target for mHCC1.
Conclusion: Our study highlighted mechanistic differences among metabolic subtypes and identified potential therapeutic targets for subtype-specific treatment strategies targeting unique metabolic vulnerabilities. The immune heterogeneities across metabolic subtypes may help further clarify the association between metabolism and immune environment and guide the development of novel strategies through targeting both unique metabolic vulnerabilities and immunosuppressive triggers.

Keywords:  PPT1; differential gene expression; hepatocellular carcinoma; metabolic subtype; metabolism; personalized treatment

DOI:  https://doi.org/10.3389/fimmu.2023.1076587
Chin Med J (Engl). 2023 Apr 04.

IGFBP-3 promotes cachexia-associated lipid loss by suppressing insulin-like growth factor/insulin signaling.

Xiaohui Wang, Jia Li, Wei Zhang, Feng Wang, Yunzi Wu, Yulin Guo, Dong Wang, Xinfeng Yu, Ang Li, Fei Li, Yibin Xie.

BACKGROUND: Progressive lipid loss of adipose tissue is a major feature of cancer-associated cachexia. In addition to systemic immune/inflammatory effects in response to tumor progression, tumor-secreted cachectic ligands also play essential roles in tumor-induced lipid loss. However, the mechanisms of tumor-adipose tissue interaction in lipid homeostasis are not fully understood.
METHODS: The yki-gut tumors were induced in fruit flies. Lipid metabolic assays were performed to investigate the lipolysis level of different types of insulin-like growth factor binding protein-3 (IGFBP-3) treated cells. Immunoblotting was used to display phenotypes of tumor cells and adipocytes. Quantitative polymerase chain reaction (qPCR) analysis was carried out to examine the gene expression levels such as Acc1, Acly, and Fasn et al .
RESULTS: In this study, we revealed that tumor-derived IGFBP-3 was an important ligand directly causing lipid loss in matured adipocytes. We uncovered that IGFBP-3, which is highly expressed in cachectic tumor cells, antagonized insulin/IGF-like signaling (IIS) and impaired the balance between lipolysis and lipogenesis in 3T3-L1 adipocytes. Conditioned medium from cachectic tumor cells, such as Capan-1 and C26 cells, contained excessive IGFBP-3 that potently induced lipolysis in adipocyted. Notably, neutralization of IGFBP-3 by neutralizing antibody in the conditioned medium of cachectic tumor cells significantly alleviated the lipolytic effect and restored lipid storage in adipocytes. Furthermore, cachectic tumor cells were resistant to IGFBP-3 inhibition of IIS, ensuring their escape from IGFBP-3-associated growth suppression. Finally, cachectic tumor-derived ImpL2, the IGFBP-3 homolog, also impaired lipid homeostasis of host cells in an established cancer-cachexia model in Drosophila. Most importantly, IGFBP-3 was highly expressed in cancer tissues in pancreatic and colorectal cancer patients, especially higher in the sera of cachectic cancer patients than non-cachexia cancer patients.
CONCLUSION: Our study demonstrates that tumor-derived IGFBP-3 plays a critical role in cachexia-associated lipid loss and could be a biomarker for diagnosis of cachexia in cancer patients.

DOI: https://doi.org/10.1097/CM9.0000000000002628
Exp Ther Med. 2023 Apr;25(4): 169

IGFBP‑rP1 affects the proliferation, apoptosis and macrophage polarization of endometrial cancer cells by regulating the PI3K/AKT pathway.

Juan Gao, Shiqi Suo, Jingxia Li, Chunxia Wang, Ranran Deng, Yuxia Hu, Chunzheng Zhang.

  Insulin-like growth factor binding protein-related protein 1 (IGFBP-rP1) is a potential tumor suppressor gene in a variety of cancers including colorectal cancer and breast cancer. However, its role and the potential mechanism in endometrial carcinoma (EC) are still unclear. The purpose of this study was to explore the effect of IGFBP-rP1 on EC cell proliferation and apoptosis and its underlying mechanism. Western blot analysis and reverse transcription-quantitative PCR were used to assess protein and gene expression levels of IGFBP-rP1 in EC cells. Overexpression of IGFBP-rP1 and/or AKT serine/threonine kinase was used to analyze their effects on cell proliferation and apoptosis of the EC cells. Co-immunoprecipitation and glutathione S transferase pull-down assays were used to analyze the interaction between IGFBP-rP1 and AKT. The expression of IGFBP-rP1 in EC cells was downregulated. Overexpression of IGFBP-rP1 inhibited the proliferation and induced apoptosis of EC cells, which were abolished by the overexpression of AKT. In addition, IGFBP-rP1 directly interacted with AKT to inhibit PI3K/AKT signaling. Additionally, M0 macrophages were induced by EC cells to differentiate into M2 macrophages, which was reversed by IGFBP-rP1. Overexpression of AKT in EC cells abolished the inhibitory effect of IGFBP-rP1 on M2 polarization. IGFBP-rP1 as an oncogenic factor inhibits M2 polarization of TAMs through PI3K/AKT signaling pathway and may be a potentially valuable target for EC therapy.

Keywords:  M2 macrophages; endometrial cancer; insulin-like growth factor binding protein-related protein 1; tumor microenvironment

DOI:  https://doi.org/10.3892/etm.2023.11868