bims-rimeca 2022-03-27 papers

bims-rimeca

Biomed News

on RNA methylation in cancer

Issue of 2022–03–27
25 papers selected by
Sk Ramiz Islam, Saha Institute of Nuclear Physics

METTL14-mediated N6-methyladenosine modification of ITGB4 mRNA inhibits metastasis of clear cell renal cell carcinoma.
Fat mass and obesity associated (FTO)-mediated N6-methyladenosine modification of Krüppel-like factor 3 (KLF3) promotes osteosarcoma progression.
m6Aexpress-Reader: prediction of m6A regulated expression genes by integrating m6A sites and reader binding information in specific- context.
Mechanisms of substrate recognition and N6-methyladenosine demethylation revealed by crystal structures of ALKBH5-RNA complexes.
Lactylation-driven METTL3-mediated RNA m6A modification promotes immunosuppression of tumor-infiltrating myeloid cells.
Targeted manipulation of m6A RNA modification through CRISPR-Cas-based strategies.
ALKBH5 ameliorated liver fibrosis and suppressed HSCs activation via triggering PTCH1 activation in an m6A dependent manner.
m6A mRNA modification maintains colonic epithelial cell homeostasis via NF-κB-mediated antiapoptotic pathway.
N6-Methyladenosine Modification Participates in the Progression of Hepatitis B Virus-Related Liver Fibrosis by Regulating Immune Cell Infiltration.
SPI1-induced downregulation of FTO promotes GBM progression by regulating pri-miR-10a processing in an m6A-dependent manner.
Identification and Validation of N6-Methyladenosine-Related Biomarkers for Bladder Cancer: Implications for Immunotherapy.
Identification of N6-Methyladenosine-Related lncRNAs as a Prognostic Signature in Glioma.
YTHDF1 upregulation mediates hypoxia-dependent breast cancer growth and metastasis through regulating PKM2 to affect glycolysis.
m6A demethylase ALKBH5 promotes tumor cell proliferation by destabilizing IGF2BPs target genes and worsens the prognosis of patients with non-small-cell lung cancer.
The N6-methyladenosine modification of circALG1 promotes the metastasis of colorectal cancer mediated by the miR-342-5p/PGF signalling pathway.
Exploring m6A and m5C Epitranscriptomes upon Viral Infection: an Example with HIV.
The XRN1-regulated RNA helicase activity of YTHDC2 ensures mouse fertility independently of m6A recognition.
The molecular mechanism of METTL3 promoting the malignant progression of lung cancer.
IGF2BP2 promotes gastric cancer progression by regulating the IGF1R-RhoA-ROCK signaling pathway.
Sequencing-free Analysis of Multiple Methylations on Gene-Specific mRNAs.
m6A Methylases Regulate Myoblast Proliferation, Apoptosis and Differentiation.
MTA1-mediated RNA m6 A modification regulates autophagy and is required for infection of the rice blast fungus.
Sperm associated antigen 4 promotes SREBP1-mediated de novo lipogenesis via interaction with lamin A/C and contributes to tumor progression in hepatocellular carcinoma.
The Endoplasmic Reticulum-Stressed Head and Neck Squamous Cell Carcinoma Cells Induced Exosomal miR-424-5p Inhibits Angiogenesis and Migration of Humanumbilical Vein Endothelial Cells Through LAMC1-Mediated Wnt/β-Catenin Signaling Pathway.
Histone methyltransferase KMT2C plays an oncogenic role in prostate cancer.

Cell Commun Signal. 2022 Mar 19. 20(1): 36

METTL14-mediated N6-methyladenosine modification of ITGB4 mRNA inhibits metastasis of clear cell renal cell carcinoma.

Zhuonan Liu, Tianshui Sun, Chiyuan Piao, Zhe Zhang, Chuize Kong.

   BACKGROUND: Integrin β4 (ITGB4) participates in tumorigenesis and progression of several malignancies, but its role and related mechanisms in clear cell renal cell carcinoma (ccRCC) remain unclear.
METHODS: Quantitative real-time PCR (qRT-PCR), western blot and immunohistochemistry were used to detect mRNA and protein levels of relevant genes. Biological functions of ITGB4 and methyltransferase-like 14 (METTL14) were determined by in vitro and in vivo experiments. The levels of N6-methyladenosine (m6A) in ccRCC tissues and adjacent normal tissues were calculated via total RNA m6A quantification assay. The m6A modification of ITGB4 was demonstrated via m6A RNA immunoprecipitation (MeRIP), RIP and luciferase reporter assays.
RESULTS: ITGB4 was significantly overexpressed in ccRCC tissues and high level of ITGB4 predicted poor prognosis as well as metastasis. Functionally, ITGB4 stimulated ccRCC cell migration and invasion in vitro and metastasis in vivo with epithelial-mesenchymal transition (EMT) strengthened. Mechanically, the total levels of m6A were reduced in ccRCC tissues. METTL14, a favorable factor for ccRCC patients' prognosis, facilitated m6A modification on ITGB4 3'UTR and subsequently accelerated ITGB4 mRNA degradation, leading to its declined expression. Furthermore, the METTL14-mediated inhibition of ITGB4 expression was dependent on the YTH domain family protein 2 (YTHDF2), which acted as an m6A reader to bind to ITGB4 mRNA and to promote its decay. In addition, we demonstrated that knockdown of METTL14 promoted ccRCC cell migration, invasiveness and metastasis as well as stimulating the EMT process and the PI3K/AKT signal by overexpressing ITGB4.
CONCLUSION: Our study reveals that METTL14 inhibits ITGB4 expression via m6A modification to attenuate metastasis and EMT of ccRCC cells, suggesting the METTL14/ITGB4 axis as a potential prognostic biomarker and therapeutic target for ccRCC. Video Abstract.

Keywords:  EMT; ITGB4; Invasion; METTL14; Metastasis; Migration; YTHDF2; ccRCC; m6A

DOI:  https://doi.org/10.1186/s12964-022-00831-5
Bioengineered. 2022 Apr;13(4): 8038-8050

Fat mass and obesity associated (FTO)-mediated N6-methyladenosine modification of Krüppel-like factor 3 (KLF3) promotes osteosarcoma progression.

Hong-Jian Shan, Wen-Xiang Gu, Gang Duan, Hong-Liang Chen.

  ARSTRACTN6-methyladenosine (m6A) methylation is the most common and abundant methylation modification of eukaryotic mRNAs, which is involved in tumor initiation and progression. The study aims to explore the potential role and the regulatory mechanism of fat mass and obesity associated (FTO) in osteosarcoma (OS) progression. In this study, we detected the expressions of Krüppel-like factor 3 (KLF3) in OS cells and tissues and found that the mRNA and protein levels of KLF3 were increased in OS cells and tissues and significantly related to tumor size, metastasis, and TNM stage and poor prognosis of OS patients. FTO promoted the proliferation and invasion and suppressed apoptosis of OS cells through cell experiments in vitro. Further mechanism dissection revealed that FTO and YTHDF2 enforced the decay of KLF3 mRNA and decreased its expression. FTO-mediated mRNA demethylation inhibited KLF3 expression in the YTHDF2-dependent manner. Moreover, KLF3 overexpression abrogated FTO-induced oncogenic effects on the proliferation and invasion of OS cells. Overall, our findings showed that FTO-mediated m6A modification of KLF3 promoted OS progression, which may provide a therapeutic target for OS.

Keywords:  FTO; KLF3; m6A methylation; osteosarcoma

DOI:  https://doi.org/10.1080/21655979.2022.2051785
Methods. 2022 Mar 18. pii: S1046-2023(22)00075-5. [Epub ahead of print]

m6Aexpress-Reader: prediction of m6A regulated expression genes by integrating m6A sites and reader binding information in specific- context.

Teng Zhang, Shao-Wu Zhang, Song-Yao Zhang, Yufei Huang.

  N6-methyladenosine (m6A) is the most abundant form of mRNA modification and plays an important role in regulating gene expression. However, the mechanisms of m6A regulated gene expression in cell or condition specific, are still poorly understood. Even though, some methods are able to predict m6A regulated expression (m6A-reg-exp) genes in specific context, they don't introduce the m6A reader binding information, while this information can help to predict m6A-reg-exp genes and more clearly to explain the mechanisms of m6A-mediated gene expression process. Thus, by integrating m6A sites and reader binding information, we proposed a novel method (called m6Aexpress-Reader) to predict m6A-reg-exp genes from limited MeRIP-seq data in specific context. m6Aexpress-Reader adopts the reader binding signal strength to weight the posterior distribution of the estimated regulatory coefficients for enhancing the prediction power. By using m6Aexpress-Reader, we found the complex characteristic of m6A on gene expression regulation and the distinct regulated pattern of m6A-reg-exp genes with different reader binding. m6A readers, YTHDF2 or IGF2BP1/3 all play an important role in various cancers and the key cancer pathways. In addition, m6Aexpress-Reader reveals the distinct m6A regulated mode of reader targeted genes in cancer. m6Aexpress-Reader could be a useful tool for studying the m6A regulation on reader target genes in specific context and it can be freely accessible at: https://github.com/NWPU-903PR/m6AexpressReader.

Keywords:  Bayesian hierarchical model; m(6)A reader; m(6)A regulated gene expression; methylation level

DOI:  https://doi.org/10.1016/j.ymeth.2022.03.008
Nucleic Acids Res. 2022 Mar 25. pii: gkac195. [Epub ahead of print]

Mechanisms of substrate recognition and N6-methyladenosine demethylation revealed by crystal structures of ALKBH5-RNA complexes.

Simranjeet Kaur, Nok Yin Tam, Michael A McDonough, Christopher J Schofield, Wei Shen Aik.

AlkB homologue 5 (ALKBH5) is a ferrous iron and 2-oxoglutarate dependent oxygenase that demethylates RNA N6-methyladenosine (m6A), a post-transcriptional RNA modification with an emerging set of regulatory roles. Along with the fat mass and obesity-associated protein (FTO), ALKBH5 is one of only two identified human m6A RNA oxidizing enzymes and is a potential target for cancer treatment. Unlike FTO, ALKBH5 efficiently catalyzes fragmentation of its proposed nascent hemiaminal intermediate to give formaldehyde and a demethylated nucleoside. A detailed analysis of the molecular mechanisms used by ALKBH5 for substrate recognition and m6A demethylation is lacking. We report three crystal structures of ALKBH5 in complex with an m6A-ssRNA 8-mer substrate and supporting biochemical analyses. Strikingly, the single-stranded RNA substrate binds to the active site of ALKBH5 in a 5'-3' orientation that is opposite to single-stranded or double-stranded DNA substrates observed for other AlkB subfamily members, including single-stranded DNA bound to FTO. The combined structural and biochemical results provide insight into the preference of ALKBH5 for substrates containing a (A/G)m6AC consensus sequence motif. The results support a mechanism involving formation of an m6A hemiaminal intermediate, followed by efficient ALKBH5 catalyzed demethylation, enabled by a proton shuttle network involving Lys132 and Tyr139.

DOI: https://doi.org/10.1093/nar/gkac195
Mol Cell. 2022 Mar 16. pii: S1097-2765(22)00207-6. [Epub ahead of print]

Lactylation-driven METTL3-mediated RNA m6A modification promotes immunosuppression of tumor-infiltrating myeloid cells.

Jia Xiong, Jia He, Jun Zhu, Jiongli Pan, Weijie Liao, Hongying Ye, Haofei Wang, Yinjing Song, Yue Du, Bijun Cui, Maoguang Xue, Wanling Zheng, Xiangxing Kong, Kai Jiang, Kefeng Ding, Lihua Lai, Qingqing Wang.

  Tumor-infiltrating myeloid cells (TIMs) are crucial cell populations involved in tumor immune escape, and their functions are regulated by multiple epigenetic mechanisms. The precise regulation mode of RNA N6-methyladenosine (m6A) modification in controlling TIM function is still poorly understood. Our study revealed that the increased expression of methyltransferase-like 3 (METTL3) in TIMs was correlated with the poor prognosis of colon cancer patients, and myeloid deficiency of METTL3 attenuated tumor growth in mice. METTL3 mediated m6A modification on Jak1 mRNA in TIMs, the m6A-YTHDF1 axis enhanced JAK1 protein translation efficiency and subsequent phosphorylation of STAT3. Lactate accumulated in tumor microenvironment potently induced METTL3 upregulation in TIMs via H3K18 lactylation. Interestingly, we identified two lactylation modification sites in the zinc-finger domain of METTL3, which was essential for METTL3 to capture target RNA. Our results emphasize the importance of lactylation-driven METTL3-mediated RNA m6A modification for promoting the immunosuppressive capacity of TIMs.

Keywords:  JAK-STAT; METTL3; lactate; lactylation; m(6)A; tumor-infiltrating myeloid cells

DOI:  https://doi.org/10.1016/j.molcel.2022.02.033
Methods. 2022 Mar 17. pii: S1046-2023(22)00073-1. [Epub ahead of print]

Targeted manipulation of m6A RNA modification through CRISPR-Cas-based strategies.

Xiang Sun, Dan Ohtan Wang, Jinkai Wang.

  N6-methyladenosine (m6A) is a reversible and prevalent internal modification in RNAs and can be dynamically modulated by methyltransferase and demethylase. Targeted manipulation of m6A RNA modification is critical in studying the functions of specific m6A sites as well as developing molecular therapies through targeting m6A. The CRISPR-Cas systems including CRISPR-Cas9 and CRISPR-Cas13 have been widely used to edit and modify specific nucleotides on DNA and RNA through fusing effective proteins such as enzymes with Cas9/13. Through taking advantage of the m6A methyltransferase and demethylase, a series of CRISPR-Cas-based methods have also been developed to manipulate the m6A methylation at specific RNA sites. This review summarizes the latest CRISPR-Cas13 and Cas9 toolkits for m6A site-specific manipulation, including fundamental components, on-target efficiency, editing window, PAM/PFS requirement, and subcellularly localized targeting as well as potential limitations. We thus aim to provide an overview to assist researchers to choose an optimal tool to manipulate m6A for different purposes and also point out possible optimization strategies.

Keywords:  CRISPR-Cas13; CRISPR-Cas9; N(6)-methyladenosine; Targeted manipulation

DOI:  https://doi.org/10.1016/j.ymeth.2022.03.006
Eur J Pharmacol. 2022 Mar 19. pii: S0014-2999(22)00161-3. [Epub ahead of print] 174900

ALKBH5 ameliorated liver fibrosis and suppressed HSCs activation via triggering PTCH1 activation in an m6A dependent manner.

Jing-Jing Yang, Juan Wang, Yang Yang, Yan Yang, Jun Li, Dong Lu, Chao Lu.

  N6-methyladenosine (m6A) is the reversible epigenetic modification of mRNA biogenesis. However, its potential role in HSCs activation and liver fibrosis remains poorly understood. Here we report m6A RNA modification serves as a key layer of HSCs activation and liver fibrosis. The effects of m6A demethylase ALKBH5 on the HSCs activation and liver fibrosis were detected by loss-of-function and gain-of-function analyses. A combination of in vitro and in vivo models, including HSCs and clinical cases or CCl4-induced mice liver fibrosis, was performed to identify the regulation and function of ALKBH5 in liver fibrosis and HSCs activation. Here, we show that the level of ALKBH5 and PTCH1 was decreased in fibrosis livers; however, genetic over expression of LV5-ALKBH5 substantially reduced α-SMA and type I of collagen levels, collagen accumulation, and interstitial fibrosis, while significantly increased PTCH1 levels. Interestingly, the expression of ALKBH5 and PTCH1 was decreased in HSCs treated by TGF-β1. Moreover, over expression of ALKBH5 reduced HSCs proliferation and migration, whereas ALKBH5 knockdown facilitated HSCs proliferation and migration. Mechanistically, ALKBH5 mediated PTCH1 activation via a m6A-dependent manner. PTCH1 upregulation resulted in the hedgehog signaling inactivation, which inhibited HSCs activation. These findings indicated that ALKBH5 ameliorated liver fibrosis and suppressed HSCs activation via triggering PTCH1 activation in a m6A dependent manner, and provides insight into critical roles of m6A methylation in liver fibrosis.

Keywords:  ALKBH5; Hedgehog; Hepatic stellate cells; Liver fibrosis; Migration; PTCH1

DOI:  https://doi.org/10.1016/j.ejphar.2022.174900
Sci Adv. 2022 Mar 25. 8(12): eabl5723

m6A mRNA modification maintains colonic epithelial cell homeostasis via NF-κB-mediated antiapoptotic pathway.

Ting Zhang, Chenbo Ding, Huifang Chen, Jun Zhao, Zhejun Chen, Baiwen Chen, Kaiqiong Mao, Yajuan Hao, Manolis Roulis, Hao Xu, Yuval Kluger, Qiang Zou, Youqiong Ye, Meixiao Zhan, Richard A Flavell, Hua-Bing Li.

Colonic mucosal barrier dysfunction is one of the major causes of inflammatory bowel disease (IBD). However, the mechanisms underlying mucosal barrier dysfunction are poorly understood. N6-methyladenosine (m6A) mRNA modification is an important modulator of epitranscriptional regulation of gene expression, participating in multiple physiological and pathological processes. However, the function of m6A modification in colonic epithelial cells and stem cells is unknown. Here, we show that m6A modification is essential for maintaining the homeostatic self-renewal in colonic stem cells. Specific deletion of the methyltransferase 14 (Mettl14) gene in mouse colon resulted in colonic stem cell apoptosis, causing mucosal barrier dysfunction and severe colitis. Mechanistically, we revealed that Mettl14 restricted colonic epithelial cell death by regulating the stability of Nfkbia mRNA and modulating the NF-κB pathway. Our results identified a previously unidentified role for m6A modification in colonic epithelial cells and stem cells, suggesting that m6A modification may be a potential therapeutic target for IBD.

DOI: https://doi.org/10.1126/sciadv.abl5723
Front Med (Lausanne). 2022 ;9 821710

N6-Methyladenosine Modification Participates in the Progression of Hepatitis B Virus-Related Liver Fibrosis by Regulating Immune Cell Infiltration.

Tong Zhao, Jianni Qi, Tiantian Liu, Hao Wu, Qiang Zhu.

   Aim: N6-methyladenosine (m6A) modification has been demonstrated to play an important part in hepatitis B virus (HBV) infection and immune response. This study aims to further investigate whether m6A modification plays an important role in the progression of HBV-related liver fibrosis through the regulation of immune cell infiltration.
Methods: In this study, 124 chronically HBV infected cases were obtained from the Gene Expression Omnibus database. In total, 489 m6A-and-stage related genes were selected to be associated with the m6A modification and the stage of liver fibrosis. Based on these genes, we identified two distinct gene clusters, gene clusterA and gene clusterB. The immune characteristics of the two clusters were comprehensively compared. The m6A-S score was constructed as quantification of individual m6A status. The correlations between m6A regulators and infiltrating immune cells were examined and compared in different pairs of groups with various m6A traits.
Results: Biological functions, immune cell infiltration, and cytokines expression were compared between the two gene clusters proving that the gene clusterB was more immune active and had a more advanced liver fibrosis stage. The m6A-S score was associated with immune infiltration and the progression of liver fibrosis. Five different grouping conditions with different m6A traits were set up. According to the intersection of significant genes and cells, ALKBH5 interacting with macrophage and WTAP interacting with nature killer T cells may be key points in the progress of liver fibrosis.
Conclusions: N6-methyladenosine modification is closely related to the immune cell infiltration and the fibrosis stage of chronic HBV-infected liver tissue. It provides us a better understanding of the progression of liver cirrhosis via evaluating the m6A modification pattern and immune infiltration characteristics.

Keywords:  N6-methyladenosine; hepatitis B virus; immune cell infiltration; immunity; liver fibrosis

DOI:  https://doi.org/10.3389/fmed.2022.821710
Mol Ther Nucleic Acids. 2022 Mar 08. 27 699-717

SPI1-induced downregulation of FTO promotes GBM progression by regulating pri-miR-10a processing in an m6A-dependent manner.

Shouji Zhang, Shulin Zhao, Yanhua Qi, Boyan Li, Huizhi Wang, Ziwen Pan, Hao Xue, Chuandi Jin, Wei Qiu, Zihang Chen, Qindong Guo, Yang Fan, Jianye Xu, Zijie Gao, Shaobo Wang, Xing Guo, Lin Deng, Shilei Ni, Fuzhong Xue, Jian Wang, Rongrong Zhao, Gang Li.

  As one of the most common post-transcriptional modifications of mRNAs and noncoding RNAs, N6-methyladenosine (m6A) modification regulates almost every aspect of RNA metabolism. Evidence indicates that dysregulation of m6A modification and associated proteins contributes to glioblastoma (GBM) progression. However, the function of fat mass and obesity-associated protein (FTO), an m6A demethylase, has not been systematically and comprehensively explored in GBM. Here, we found that decreased FTO expression in clinical specimens correlated with higher glioma grades and poorer clinical outcomes. Functionally, FTO inhibited growth and invasion in GBM cells in vitro and in vivo. Mechanistically, FTO regulated the m6A modification of primary microRNA-10a (pri-miR-10a), which could be recognized by reader HNRNPA2B1, recruiting the microRNA microprocessor complex protein DGCR8 and mediating pri-miR-10a processing. Furthermore, the transcriptional activity of FTO was inhibited by the transcription factor SPI1, which could be specifically disrupted by the SPI1 inhibitor DB2313. Treatment with this inhibitor restored endogenous FTO expression and decreased GBM tumor burden, suggesting that FTO may serve as a novel prognostic indicator and therapeutic molecular target of GBM.

Keywords:  DB2313; FTO; N6-methyladenosine; SPI1; glioblastoma; pri-miR-10a

DOI:  https://doi.org/10.1016/j.omtn.2021.12.035
Front Oncol. 2022 ;12 820242

Identification and Validation of N6-Methyladenosine-Related Biomarkers for Bladder Cancer: Implications for Immunotherapy.

Hongyu Deng, Faqing Tang, Ming Zhou, Dongyong Shan, Xingyu Chen, Ke Cao.

  N6-methyladenosine (m6A) has emerged as one of the most important modifications of RNA. Based on the expression of 23 different modes of m6A regulatory factors, we identified three different m6A modification patterns in bladder cancer. The effects of the three different modes of m6A modification on clinicopathological characteristics, immune cell infiltration levels and expression levels of immune checkpoint genes were comprehensively analyzed. In addition, the effects of different modes of m6A modification on the therapeutic efficacy of anti-PD-L1 immunotherapy (atezolizumab) are also discussed. Our results confirm that m6A methylation plays an important role in immune cell recruitment in the tumor microenvironment of bladder cancer, which influences the efficacy of anti-PD-L1 therapy for bladder cancer. We further confirmed the important role of FTO protein in the biological function of bladder cancer cells by performing in vitro experiments. FTO functions as an oncogene in bladder cancer cells, and upon FTO knockdown, the level of m6A enzyme activity in bladder cancer cells was significantly increased, apoptosis was increased, and cell proliferation and cell invasion were reduced. In addition, our study also confirmed that K216H and K216E are probably important targets for regulating FTO. We provide new insights into the regulatory pathways of the immune microenvironment and the methylation function of m6A in bladder cancer, which will help in designing novel diagnostic methods, prognostic tools, and therapeutic targets.

Keywords:  N6-methyladenosine; anti-PD-L1; bladder cancer; immune checkpoint inhibitors; tumor immune microenvironment

DOI:  https://doi.org/10.3389/fonc.2022.820242
Front Oncol. 2022 ;12 789283

Identification of N6-Methyladenosine-Related lncRNAs as a Prognostic Signature in Glioma.

Yujia Chen, Yuduo Guo, Shenglun Li, Jiacheng Xu, Xiang Wang, Weihai Ning, Lixin Ma, Yanming Qu, Mingshan Zhang, Hongwei Zhang.

  N6-methyladenosine (m6A) modification is the most abundant modification in long noncoding RNAs (lncRNAs). Current studies have shown that the abnormal expression of m6A-related genes is closely associated with the tumorigenesis and progression of glioma. However, the role of m6A-related lncRNAs in glioma development is still unclear. Herein, we screened 566 m6A-related lncRNAs in glioma from The Cancer Genome Atlas (TCGA) database. The expression pattern of these lncRNAs could cluster samples into two groups, in which various classical tumor-related functions and the tumor immune microenvironment were significantly different. Subsequently, a nine-factor m6A-related lncRNA prognostic signature (MLPS) was constructed by using a LASSO regression analysis in the training set and was validated in the test set and independent datasets. The AUC values of the MLPS were 0.881, 0.918 and 0.887 for 1-, 3- and 5-year survival in the training set, respectively, and 0.856, 0.916 and 0.909 for 1-, 3-, and 5-year survival in the test set, respectively. Stratification analyses of the MLPS illustrated its prognostic performance in gliomas with different characteristics. Correlation analyses showed that the infiltrations of monocytes and tumor-associated macrophages (TAMs) were significantly relevant to the risk score in the MLPS. Moreover, we detected the expression of four MLPS factors with defined sequences in glioma and normal cells by using RT-PCR. Afterwards, we investigated the functions of LNCTAM34A (one of the MLPS factors) in glioma cells, which have rarely been reported. Via in vitro experiments, LNCTAM34A was demonstrated to promote the proliferation, migration and epithelial-mesenchymal transition (EMT) of glioma cells. Overall, our study revealed the critical role of m6A-related lncRNAs in glioma and elucidated that LNCTAM34A could promote glioma proliferation, migration and EMT.

Keywords:  N6-methyladenosine; The Cancer Genome Atlas; glioma; long non-coding RNA; prognostic signature

DOI:  https://doi.org/10.3389/fonc.2022.789283
Cell Death Dis. 2022 Mar 23. 13(3): 258

YTHDF1 upregulation mediates hypoxia-dependent breast cancer growth and metastasis through regulating PKM2 to affect glycolysis.

Xuemei Yao, Wei Li, Liqi Li, Menghuan Li, Youbo Zhao, De Fang, Xiaohua Zeng, Zhong Luo.

N6-methyladenosine modification is the most common RNA modification mechanism in mammals. YTHDF1, a m6A reader, can recognize the m6A of mRNAs to facilitate the interaction with the mRNA ribosome assembly and recruitment of translation initiators to promote translation. From a clinical perspective, YTHDF1 upregulation is frequently observed in breast cancer, but its involvement in those cancer-related events is still unclear. Here we report that YTHDF1 is a cancer driver capable of facilitating the proliferation and invasion of breast cancer cells as well as enhancing tumorigenicity and metastasis through promoting glycolysis. We found that tumor hypoxia can transcriptionally induce HIF1α and post-transcriptionally inhibit the expression of miR-16-5p to promote YTHDF1 expression, which could sequentially enhance tumor glycolysis by upregulating PKM2 and eventually increase the tumorigenesis and metastasis potential of breast cancer cells. Inhibiting YTHDF1 via gene knockdown or miR-16-5p would significantly abolish YTHDF1-dependent tumor growth and metastasis. In summary, we identified the role of the YTHDF1-PKM2 signal axis in the occurrence and development of breast cancer, which can be used as a potential target for breast cancer treatment.

DOI: https://doi.org/10.1038/s41419-022-04711-1
Cancer Gene Ther. 2022 Mar 22.

m6A demethylase ALKBH5 promotes tumor cell proliferation by destabilizing IGF2BPs target genes and worsens the prognosis of patients with non-small-cell lung cancer.

Kazuo Tsuchiya, Katsuhiro Yoshimura, Yuji Iwashita, Yusuke Inoue, Tsutomu Ohta, Hirofumi Watanabe, Hidetaka Yamada, Akikazu Kawase, Masayuki Tanahashi, Hiroshi Ogawa, Kazuhito Funai, Kazuya Shinmura, Takafumi Suda, Haruhiko Sugimura.

The modification of N6-methyladenosine (m6A) in RNA and its eraser ALKBH5, an m6A demethylase, play an important role across various steps of human carcinogenesis. However, the involvement of ALKBH5 in non-small-cell lung cancer (NSCLC) development remains to be completely elucidated. The current study revealed that the expression of ALKBH5 was increased in NSCLC and increased expression of ALKBH5 worsened the prognosis of patients with NSCLC. In vitro study revealed that ALKBH5 knockdown suppressed cell proliferation ability of PC9 and A549 cells and promoted G1 arrest and increased the number of apoptotic cells. Furthermore, ALKBH5 overexpression increased the cell proliferation ability of the immortalized cell lines. Microarray analysis and western blotting revealed that the expression of CDKN1A (p21) or TIMP3 was increased by ALKBH5 knockdown. These alterations were offset by a double knockdown of both ALKBH5 and one of the IGF2BPs. The decline of mRNAs was, at least partly, owing to the destabilization of these mRNAs by one of the IGF2BPs. In conclusions, the ALKBH5-IGF2BPs axis promotes cell proliferation and tumorigenicity, which in turn causes the unfavorable prognosis of NSCLC.

DOI: https://doi.org/10.1038/s41417-022-00451-8
Mol Cancer. 2022 Mar 19. 21(1): 80

The N6-methyladenosine modification of circALG1 promotes the metastasis of colorectal cancer mediated by the miR-342-5p/PGF signalling pathway.

Changwei Lin, Min Ma, Yi Zhang, Liang Li, Fei Long, Canbin Xie, Hua Xiao, Teng Liu, Buning Tian, Kaiyan Yang, Yihang Guo, Miao Chen, Jin Chou, Ni Gong, Xiaorong Li, Gui Hu.

   BACKGROUND: Previous studies have shown that the N6-methyladenosine (m6A) modification enhances the binding ability of mRNAs/long noncoding RNAs (lncRNAs) to microRNAs (miRNAs), but the impact of this modification on the competitive endogenous RNA (ceRNA) function of circular RNAs (circRNAs) is unclear.
METHODS: We used a human circRNA microarray to detect the expression profiles of circRNAs in 3 pairs of cancer and paracancerous tissues from patients with colorectal cancer (CRC) and 3 pairs of peripheral blood specimens from patients with CRC and healthy individuals. The circRNAs highly expressed in both peripheral blood and tumour tissues of patients with CRC, including circALG1, were screened. A quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analysis of an expanded sample size was performed to detect the expression level of circALG1 in peripheral blood and tumour tissues of patients with CRC and determine its correlation with clinicopathological features, and circRNA loop-forming validation and stability assays were then conducted. Transwell assays and a nude mouse cancer metastasis model were used to study the function of circALG1 in CRC and the role of altered m6A modification levels on the regulation of circALG1 function. qRT-PCR, western blot (WB), Transwell, RNA-binding protein immunoprecipitation (RIP), RNA antisense purification (RAP), and dual-luciferase reporter gene assays were performed to analyse the ceRNA mechanism of circALG1 and the effect of the m6A modification of circALG1 on the ceRNA function of this circRNA.
RESULTS: CircALG1 was highly expressed in both the peripheral blood and tumour tissues of patients with CRC and was closely associated with CRC metastasis. CircALG1 overexpression promoted the migration and invasion of CRC cells, and circALG1 silencing and reduction of the circALG1 m6A modification level inhibited CRC cell migration and invasion. In vivo experiments further confirmed the prometastatic role of circALG1 in CRC. Further mechanistic studies showed that circALG1 upregulated the expression of placental growth factor (PGF) by binding to miR-342-5p and that m6A modification enhanced the binding of circALG1 to miR-342-5p and promoted its ceRNA function.
CONCLUSION: M6A modification enhances the binding ability of circALG1 to miR-342-5p to promote the ceRNA function of circALG1, and circALG1 could be a potential therapeutic target in and a prognostic marker for CRC.

Keywords:  Colorectal cancer; Competitive endogenous RNA; N6-methyladenosine modification; Placental growth factor; circALG1; miR-342-5p

DOI:  https://doi.org/10.1186/s12943-022-01560-6
J Vis Exp. 2022 Mar 05.

Exploring m6A and m5C Epitranscriptomes upon Viral Infection: an Example with HIV.

Sara Cristinelli, Paolo Angelino, Angela Ciuffi.

The role of RNA modifications in biological processes has been the focus of an increasing number of studies in the last few years and is known nowadays as epitranscriptomics. Among others, N6-methyladenosine (m6A) and 5-methylcytosine (m5C) RNA modifications have been described on mRNA molecules and may have a role in modulating cellular processes. Epitranscriptomics is thus a new layer of regulation that must be considered in addition to transcriptomic analyses, as it can also be altered or modulated by exposure to any chemical or biological agent, including viral infections. Here, we present a workflow that allows analysis of the joint cellular and viral epitranscriptomic landscape of the m6A and m5C marks simultaneously, in cells infected or not with the human immunodeficiency virus (HIV). Upon mRNA isolation and fragmentation from HIV- infected and non-infected cells, we used two different procedures: MeRIP-Seq, an RNA immunoprecipitation-based technique, to enrich for RNA fragments containing the m6A mark and BS-Seq, a bisulfite conversion-based technique, to identify the m5C mark at a single nucleotide resolution. Upon methylation-specific capture, RNA libraries are prepared for high-throughput sequencing. We also developed a dedicated bioinformatics pipeline to identify differentially methylated (DM) transcripts independently from their basal expression profile. Overall, the methodology allows exploration of multiple epitranscriptomic marks simultaneously and provides an atlas of DM transcripts upon viral infection or any other cell perturbation. This approach offers new opportunities to identify novel players and novel mechanisms of cell response, such as cellular factors promoting or restricting viral replication.

DOI: https://doi.org/10.3791/62426
Mol Cell. 2022 Mar 10. pii: S1097-2765(22)00208-8. [Epub ahead of print]

The XRN1-regulated RNA helicase activity of YTHDC2 ensures mouse fertility independently of m6A recognition.

Lingyun Li, Kyrylo Krasnykov, David Homolka, Pascal Gos, Mateusz Mendel, Richard J Fish, Radha Raman Pandey, Ramesh S Pillai.

  The functional consequence of N6-methyladenosine (m6A) RNA modification is mediated by "reader" proteins of the YTH family. YTH domain-containing 2 (YTHDC2) is essential for mammalian fertility, but its molecular function is poorly understood. Here, we identify U-rich motifs as binding sites of YTHDC2 on 3' UTRs of mouse testicular RNA targets. Although its YTH domain is an m6A-binder in vitro, the YTH point mutant mice are fertile. Significantly, the loss of its 3'→5' RNA helicase activity causes mouse infertility, with the catalytic-dead mutation being dominant negative. Biochemical studies reveal that the weak helicase activity of YTHDC2 is enhanced by its interaction with the 5'→3' exoribonuclease XRN1. Single-cell transcriptomics indicate that Ythdc2 mutant mitotic germ cells transition into meiosis but accumulate a transcriptome with mixed mitotic/meiotic identity that fail to progress further into meiosis. Finally, our demonstration that ythdc2 mutant zebrafish are infertile highlights its conserved role in animal germ cell development.

Keywords:  DExH helicase; MEIOC; RBM46; RNA helicase; XRN1; YTH; YTHDC2; m(6)A; oogenesis; spermatogenesis

DOI:  https://doi.org/10.1016/j.molcel.2022.02.034
Cancer Cell Int. 2022 Mar 24. 22(1): 133

The molecular mechanism of METTL3 promoting the malignant progression of lung cancer.

Chao Ma, Rui-Jie Ma, Kang Hu, Qi-Ming Zheng, Ye-Peng Wang, Nan Zhang, Zhi-Gang Sun.

  Lung cancer remains one of the major causes of cancer-related death globally. Recent studies have shown that aberrant m6A levels caused by METTL3 are involved in the malignant progression of various tumors, including lung cancer. The m6A modification, the most abundant RNA chemical modification, regulates RNA stabilization, splicing, translation, decay, and nuclear export. The methyltransferase complex plays a key role in the occurrence and development of many tumors by installing m6A modification. In this complex, METTL3 is the first identified methyltransferase, which is also the major catalytic enzyme. Recent findings have revealed that METTL3 is remarkably associated with different aspects of lung cancer progression, influencing the prognosis of patients. In this review, we will focus on the underlying mechanism of METT3 in lung cancer and predict the future work and potential clinical application of targeting METTL3 for lung cancer therapy.

Keywords:  Inhibitors; Lung cancer; METTL3; Malignant progression; Prognosis; Tumor microenvironment; m6A

DOI:  https://doi.org/10.1186/s12935-022-02539-5
Cell Signal. 2022 Mar 16. pii: S0898-6568(22)00074-2. [Epub ahead of print] 110313

IGF2BP2 promotes gastric cancer progression by regulating the IGF1R-RhoA-ROCK signaling pathway.

Dong Liu, A-Dong Xia, Le-Ping Wu, Song Li, Kai Zhang, Dan Chen.

   BACKGROUND: Our study aimed to probe the intrinsic and concrete molecular mechanism of IGF2 mRNA-binding protein 2 (IGF2BP2) in gastric cancer (GC).
METHODS: The mRNA and protein expressions were assessed using qRT-PCR and western blot, respectively. CCK-8 assay was employed to determine cell proliferation. Levels of TNFα and IL-1β were analyzed using ELISA. Furthermore, cell apoptosis was evaluated using flow cytometry analysis. Cell migration and invasion were evaluated using Transwell assay. The experiment of tumor formation in nude mice was employed to analyze the effect of IGF2BP2 in regulating GC tumor growth and lung metastasis in vivo. Finally, the binding relationship between IGF1R and IGF2BP2 was verified using RIP and RNA pull down assays.
RESULTS: IGF2BP2 was significantly elevated in both GC tissues and cells. Silencing of IGF2BP2 dramatically suppressed the inflammation, proliferation, migration and invasion, yet promoted cell apoptosis in vitro and in vivo. Furthermore, IGF2BP2, as a m6A reader, was proved to increase the expression of IGF1R by identifying m6A methylation modification sites in IGF1R mRNA, thus activating RhoA-ROCK pathway. Importantly, the anti-carcinogenic impacts of IGFBP2 silence were restrained by IGF1R overexpression, which was eliminated by the inactivation of RhoA-ROCK.
CONCLUSION: We emphasized the oncogenic role of IGF2BP2 in gastric carcinogenesis and confirmed its activation is partly due to the activation of IGF1R-RhoA-ROCK signaling pathway. Our findings identified that IGF2BP2 might be a promising prognostic biomarker and provided clinical translational potential.

Keywords:  Gastric cancer; IGF1R; IGF2BP2; RhoA-ROCK pathway

DOI:  https://doi.org/10.1016/j.cellsig.2022.110313
J Am Chem Soc. 2022 Mar 23.

Sequencing-free Analysis of Multiple Methylations on Gene-Specific mRNAs.

Xi Zhao, Xianglin Ji, Jin Qu, Kai Xie, Zixun Wang, Peilin Fang, Yuan Wang, Youyang Wan, Yang Yang, Wenjun Zhang, Peng Shi.

RNA epigenetics is a new layer of mechanism to regulate gene expression, but limited techniques are available to profile the status of mRNA modifications. Here, we describe a molecule proximity-based technique for simultaneous analysis of multiple types of mRNA methylation with specific gene information in living cells. N6-methyladenosine (m6A) or N1-methyladenosine (m1A) modifications on multiple mRNAs can be individually or simultaneously analyzed. A chip fabricated with vertically aligned, high-aspect-ratio diamond nanoneedles was used to access the intracellular domain in a minimum-invasive format and to isolate the mRNAs out of the cell cytoplasm while keeping cells alive. In the subsequent on-chip analytical procedures, the isolated RNAs were encoded, amplified, and visualized to derive a quantitative measurement of the associated gene-specific m6A or m1A modifications. Notably, a proximity ligation approach was developed to resolve dual methylation on an individual mRNA segment. Using this method, we investigated the dynamics of mRNA methylation in mammalian cells under physical or chemical stimuli and showed that m6A and m1A in mRNAs are heavily involved in the cellular stress response. Our results also suggested the common existence of single m6A modification in the basigin (BSG) mRNA but a rare occurrence of m6A and m1A dual methylation in the same BSG transcript.

DOI: https://doi.org/10.1021/jacs.2c01036
Animals (Basel). 2022 Mar 18. pii: 773. [Epub ahead of print]12(6):

m6A Methylases Regulate Myoblast Proliferation, Apoptosis and Differentiation.

Xinran Yang, Chugang Mei, Xinhao Ma, Jiawei Du, Jianfang Wang, Linsen Zan.

  N6-methyladenosine (m6A) plays an important role in regulating gene expression. Previous studies found that m6A methylation affects skeletal muscle development. However, the effect of m6A methylases on bovine skeletal myogenesis is still unclear. Here, we found that the expression of m6A demethylases (FTO and ALKBH5) was significantly higher in the longissimus dorsi muscle of adult cattle than in newborn cattle. In contrast, the expression of m6A methyltransferases (METTL3, METTL14 and WTAP) was reduced. The mRNA expression of all five genes was found to be increased during the myogenesis of myoblasts in vitro. Knockdown of FTO or METTL3 promoted myoblast proliferation, inhibited myoblast apoptosis and suppressed myogenic differentiation, whereas ALKBH5 knockdown had the opposite effect. METTL14 knockdown enhanced myoblast proliferation and impaired myogenic differentiation. WTAP knockdown attenuated proliferation and contributed to apoptosis but did not affect differentiation. Furthermore, the functional domains of these five m6A methylases are conserved across species. Our results suggest that m6A methylases are involved in regulating skeletal muscle development and that there may be a complex network of m6A methylation regulating skeletal myogenesis.

Keywords:  N6-methyladenosine; apoptosis; cattle; m6A methylases; myoblast differentiation; myogenesis; proliferation

DOI:  https://doi.org/10.3390/ani12060773
New Phytol. 2022 Mar 26.

MTA1-mediated RNA m6 A modification regulates autophagy and is required for infection of the rice blast fungus.

Zhiyong Ren, Bozeng Tang, Junjie Xing, Caiyun Liu, Xuan Cai, Ahmed Hendy, Muhammad Kamran, Hao Liu, Lu Zheng, Junbing Huang, Xiao-Lin Chen.

  In eukaryotes, N6 -methyladenosine (m6 A) is abundant on mRNA, which plays key roles in the regulation of RNA function. However, the roles and regulatory mechanisms of m6 A in phytopathogenic fungi are still largely unknown. Combing with biochemical analysis, MeRIP-seq and RNAseq methods, as well as biological analysis, we showed Magnaporthe oryzae MTA1 gene is an ortholog of human METTL4, which is involved in m6 A modification and plays a critical role in autophagy for fungal infection. The Δmta1 mutant showed reduced virulence due to blockage of appressorial penetration and invasive growth. Moreover, the autophagy process was severely disordered in the mutant. MeRIP-seq identified 659 hypomethylated m6 A peaks covering 595 mRNAs in Δmta1 appressorium, 114 m6 A peaks was negatively related to mRNA abundance, including several ATG gene's transcripts. Typically, the mRNA abundance of MoATG8 was also increased in the single m6 A site mutant ∆atg8/MoATG8A982C , leading to an autophagy disorder. Our findings reveal the functional importance of the m6 A methylation in infection of M. oryzae and provide novel insight into regulatory mechanism of plant pathogenic fungi.

Keywords:   Magnaporthe oryzae ; appressorium maturation; autophagy; fungal infection; m6A methylation; m6A methyltransferase

DOI:  https://doi.org/10.1111/nph.18117
Cancer Lett. 2022 Mar 17. pii: S0304-3835(22)00125-2. [Epub ahead of print] 215642

Sperm associated antigen 4 promotes SREBP1-mediated de novo lipogenesis via interaction with lamin A/C and contributes to tumor progression in hepatocellular carcinoma.

Tengfei Liu, Junming Yu, Chao Ge, Fangyu Zhao, Jing Chen, Chunxiao Miao, Wenjiao Jin, Qingqing Zhou, Qin Geng, Hechun Lin, Hua Tian, Taoyang Chen, Haiyang Xie, Ying Cui, Ming Yao, Xiuying Xiao, Jinjun Li, Hong Li.

  Hepatocellular carcinoma (HCC) is a highly malignant tumor and its progression is associated with altered lipid metabolism in precancerous lesions, such as non-alcoholic fatty liver disease. Here, we identified sperm associated antigen 4 (SPAG4), and explored its oncogenic role in HCC progression. Database analysis and immunohistochemistry indicated increased level of SPAG4 in HCC tissues which was of prognostic value. Gain/loss-of-function experiments showed that SPAG4 exerts oncogenic roles in HCC growth both in vitro and in vivo. RNA sequencing revealed activation of a lipogenic state and SREBP1-mediated pathway following SPAG4 overexpression. Mechanistically, the N-terminal region of SPAG4 bound to lamin A/C, which increased SREBP1 expression, nuclear translocation, and transcriptional activity. Treatment with orlistat, a lipid synthesis inhibitor, reversed SPAG4-mediated oncogenic effects, and its efficacy varied with SPAG4 level. The effect of orlistat was further amplified when combined with sorafenib in tumor xenograft mouse models. Our study provides evidence that SPAG4 mediates HCC progression by affecting lipid metabolism. Administration of orlistat combined with sorafenib reverses SPAG4-mediated oncogenesis in HCC cells and ectopic xenograft tumors in mice, suggesting that this pathway represent a potential target for HCC treatment.

Keywords:  Hepatocellular carcinoma; Lamin A/C; Lipogenesis; SPAG4; SREBP1

DOI:  https://doi.org/10.1016/j.canlet.2022.215642
Cell Transplant. 2022 Jan-Dec;31:31 9636897221083549

The Endoplasmic Reticulum-Stressed Head and Neck Squamous Cell Carcinoma Cells Induced Exosomal miR-424-5p Inhibits Angiogenesis and Migration of Humanumbilical Vein Endothelial Cells Through LAMC1-Mediated Wnt/β-Catenin Signaling Pathway.

Zeyu Wang, Pengfei Jiao, Yi Zhong, Huan Ji, Yaqin Zhang, Haiyang Song, Hongming Du, Xu Ding, Heming Wu.

  Under endoplasmic reticulum (ER) stress, tumor plays multifaceted roles in endothelial cell dysfunction through secreting exosomal miRNAs. However, for the head and neck squamous cell carcinoma (HNSCC), it is still unclear about the impact of ER-stressed HNSCC cell derived exosomes on vascular endothelial cells. To address this gap, herein, systemic research was conducted including isolation and characterization of ER-stressed HNSCC cell (HN4 cell line as an in vitro model) derived exosomes, identification of regulatory exosomal miRNAs, target exploration and downstream signaling pathway investigation of exosomal miRNAs in human umbilical vein endothelial cell (HUVEC). ER-stressed HN4 cell-derived exosomes inhibited angiogenesis and migration of HUVEC cells in vitro. Furthermore, RNA-seq analysis demonstrated that miR-424-5p was highly upregulated in ER-stressed HN4 cell-derived exosomes. Through matrigel tube formation and transwell assays of HUVEC cells, miR-424-5p displayed great capabilities on inhibiting angiogenesis and migration. Finally, based on western blot and luciferase reporter, it was demonstrated that LAMC1 is the target of miR-424-5p which could inhibit the angiogenesis and migration of HUVEC cells by repressing the LAMC1-mediated Wnt/β-catenin signaling pathway. ER-stressed HNSCC cell-induced exosomal miR-424-5p inhibits angiogenesis and migration of HUVEC cells through LAMC1-mediated Wnt/β-catenin signaling pathway. This study offers a new insight for understanding the complicated mechanism behind ER-stress induced anti-angiogenesis of HNSCC.

Keywords:  Wnt/β-catenin; anti-angiogenesis; endoplasmic reticulum stress; exosomal miRNAs; head and neck squamous cell carcinoma

DOI:  https://doi.org/10.1177/09636897221083549
J Cancer Res Clin Oncol. 2022 Mar 23.

Histone methyltransferase KMT2C plays an oncogenic role in prostate cancer.

Jianpo Lian, Chengdang Xu, Xi Chen, Shengsong Huang, Denglong Wu.

   PURPOSE: Prostate cancer (PCa) is a leading cause of morbidity and mortality in males. Epigenetic modifier abnormalities are becoming a driving event in PCa. The specific role of KMT2C, a histone methyltransferase that is frequently aberrant in various tumors, is poorly understood in PCa. This study aimed to reveal the potential carcinogenic role of KMT2C in PCa.
METHODS: We first examined the expression levels of KMT2C in prostate cancer tissues. Then, we assessed the function of KMT2C in prostate cancer cell proliferation, colony formation, and migration. To explore the mechanism of the biological consequences, RNA-seq and CHIP-qPCR were performed. We also analyzed the effects of overexpression of the KMT2C downstream genes CLDN8 and ITGAV to reverse the effects of KMT2C on prostate cancer cells.
RESULTS: Herein, we first confirmed KMT2C overexpression in PCa at the transcript and protein levels. Knocking down KMT2C in VCaP and LNCaP cells inhibited cell viability, colony formation, and migration. Consistently, stable KMT2C depletion effectively decreased tumor growth by approximately 70% in vivo. Mechanistically, the results suggested that CLDN8 and ITGAV are two key downstream genes of KMT2C and further regulate the MAPK/ERK and EMT pathways.
CONCLUSION: Our study suggests that KMT2C plays an oncogenic role in PCa. One of the mechanisms may be the epigenetic regulation of CLDN8 and ITGAV by KMT2C to modulate tumor-signaling pathways. Therefore, KMT2C may serve as a potential therapeutic target for PCa patients.

Keywords:  EMT; Epigenetics; KMT2C; MAPK/ERK signaling; Prostate cancer

DOI:  https://doi.org/10.1007/s00432-022-03968-5