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      METTL3 facilitates tumor progression via an m 6A-IGF2BP2-dependent mechanism in colorectal carcinoma

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          Abstract

          Background

          Colorectal carcinoma (CRC) is one of the most common malignant tumors, and its main cause of death is tumor metastasis. RNA N 6-methyladenosine (m 6A) is an emerging regulatory mechanism for gene expression and methyltransferase-like 3 (METTL3) participates in tumor progression in several cancer types. However, its role in CRC remains unexplored.

          Methods

          Western blot, quantitative real-time PCR (RT-qPCR) and immunohistochemical (IHC) were used to detect METTL3 expression in cell lines and patient tissues. Methylated RNA immunoprecipitation sequencing (MeRIP-seq) and transcriptomic RNA sequencing (RNA-seq) were used to screen the target genes of METTL3. The biological functions of METTL3 were investigated in vitro and in vivo. RNA pull-down and RNA immunoprecipitation assays were conducted to explore the specific binding of target genes. RNA stability assay was used to detect the half-lives of the downstream genes of METTL3.

          Results

          Using TCGA database, higher METTL3 expression was found in CRC metastatic tissues and was associated with a poor prognosis. MeRIP-seq revealed that SRY (sex determining region Y)-box 2 (SOX2) was the downstream gene of METTL3. METTL3 knockdown in CRC cells drastically inhibited cell self-renewal, stem cell frequency and migration in vitro and suppressed CRC tumorigenesis and metastasis in both cell-based models and PDX models. Mechanistically, methylated SOX2 transcripts, specifically the coding sequence (CDS) regions, were subsequently recognized by the specific m 6A “reader”, insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2), to prevent SOX2 mRNA degradation. Further, SOX2 expression positively correlated with METTL3 and IGF2BP2 in CRC tissues. The combined IHC panel, including “writer”, “reader”, and “target”, exhibited a better prognostic value for CRC patients than any of these components individually.

          Conclusions

          Overall, our study revealed that METTL3, acting as an oncogene, maintained SOX2 expression through an m 6A-IGF2BP2-dependent mechanism in CRC cells, and indicated a potential biomarker panel for prognostic prediction in CRC.

          Electronic supplementary material

          The online version of this article (10.1186/s12943-019-1038-7) contains supplementary material, which is available to authorized users.

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          Most cited references16

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          Transcriptome-wide mapping of N(6)-methyladenosine by m(6)A-seq based on immunocapturing and massively parallel sequencing.

          N(6)-methyladenosine-sequencing (m(6)A-seq) is an immunocapturing approach for the unbiased transcriptome-wide localization of m(6)A in high resolution. To our knowledge, this is the first protocol to allow a global view of this ubiquitous RNA modification, and it is based on antibody-mediated enrichment of methylated RNA fragments followed by massively parallel sequencing. Building on principles of chromatin immunoprecipitation-sequencing (ChIP-seq) and methylated DNA immunoprecipitation (MeDIP), read densities of immunoprecipitated RNA relative to untreated input control are used to identify methylated sites. A consensus motif is deduced, and its distance to the point of maximal enrichment is assessed; these measures further corroborate the success of the protocol. Identified locations are intersected in turn with gene architecture to draw conclusions regarding the distribution of m(6)A between and within gene transcripts. When applied to human and mouse transcriptomes, m(6)A-seq generated comprehensive methylation profiles revealing, for the first time, tenets governing the nonrandom distribution of m(6)A. The protocol can be completed within ~9 d for four different sample pairs (each consists of an immunoprecipitation and corresponding input).
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            SOX2 controls tumour initiation and cancer stem-cell functions in squamous-cell carcinoma.

            Cancer stem cells (CSCs) have been reported in various cancers, including in skin squamous-cell carcinoma (SCC). The molecular mechanisms regulating tumour initiation and stemness are still poorly characterized. Here we find that Sox2, a transcription factor expressed in various types of embryonic and adult stem cells, was the most upregulated transcription factor in the CSCs of squamous skin tumours in mice. SOX2 is absent in normal epidermis but begins to be expressed in the vast majority of mouse and human pre-neoplastic skin tumours, and continues to be expressed in a heterogeneous manner in invasive mouse and human SCCs. In contrast to other SCCs, in which SOX2 is frequently genetically amplified, the expression of SOX2 in mouse and human skin SCCs is transcriptionally regulated. Conditional deletion of Sox2 in the mouse epidermis markedly decreases skin tumour formation after chemical-induced carcinogenesis. Using green fluorescent protein (GFP) as a reporter of Sox2 transcriptional expression (SOX2-GFP knock-in mice), we showed that SOX2-expressing cells in invasive SCC are greatly enriched in tumour-propagating cells, which further increase upon serial transplantations. Lineage ablation of SOX2-expressing cells within primary benign and malignant SCCs leads to tumour regression, consistent with the critical role of SOX2-expressing cells in tumour maintenance. Conditional Sox2 deletion in pre-existing skin papilloma and SCC leads to tumour regression and decreases the ability of cancer cells to be propagated upon transplantation into immunodeficient mice, supporting the essential role of SOX2 in regulating CSC functions. Transcriptional profiling of SOX2-GFP-expressing CSCs and of tumour epithelial cells upon Sox2 deletion uncovered a gene network regulated by SOX2 in primary tumour cells in vivo. Chromatin immunoprecipitation identified several direct SOX2 target genes controlling tumour stemness, survival, proliferation, adhesion, invasion and paraneoplastic syndrome. We demonstrate that SOX2, by marking and regulating the functions of skin tumour-initiating cells and CSCs, establishes a continuum between tumour initiation and progression in primary skin tumours.
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              m(6)A RNA methylation is regulated by microRNAs and promotes reprogramming to pluripotency.

              N(6)-methyladenosine (m(6)A) has been recently identified as a conserved epitranscriptomic modification of eukaryotic mRNAs, but its features, regulatory mechanisms, and functions in cell reprogramming are largely unknown. Here, we report m(6)A modification profiles in the mRNA transcriptomes of four cell types with different degrees of pluripotency. Comparative analysis reveals several features of m(6)A, especially gene- and cell-type-specific m(6)A mRNA modifications. We also show that microRNAs (miRNAs) regulate m(6)A modification via a sequence pairing mechanism. Manipulation of miRNA expression or sequences alters m(6)A modification levels through modulating the binding of METTL3 methyltransferase to mRNAs containing miRNA targeting sites. Increased m(6)A abundance promotes the reprogramming of mouse embryonic fibroblasts (MEFs) to pluripotent stem cells; conversely, reduced m(6)A levels impede reprogramming. Our results therefore uncover a role for miRNAs in regulating m(6)A formation of mRNAs and provide a foundation for future functional studies of m(6)A modification in cell reprogramming.
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                Author and article information

                Contributors
                86-20-87342280 , juhq@sysucc.org.cn
                86-20-87343333 , xurh@sysucc.org.cn
                Journal
                Mol Cancer
                Mol. Cancer
                Molecular Cancer
                BioMed Central (London )
                1476-4598
                24 June 2019
                24 June 2019
                2019
                : 18
                : 112
                Affiliations
                [1 ]ISNI 0000 0004 1803 6191, GRID grid.488530.2, State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, , Sun Yat-sen University Cancer Center, ; 651 Dongfeng East Road, Guangzhou, 510060 People’s Republic of China
                [2 ]ISNI 0000 0004 1803 6191, GRID grid.488530.2, Department of Medical Oncology, , Sun Yat-sen University Cancer Center, ; Guangzhou, 510060 China
                [3 ]ISNI 0000 0004 1803 6191, GRID grid.488530.2, Department of Pathology, , Sun Yat-sen University Cancer Center, ; Guangzhou, 510060 China
                [4 ]ISNI 0000 0004 1762 1794, GRID grid.412558.f, Department of Medical Oncology and Guangdong Key Laboratory of Liver Disease, , the Third Affiliated Hospital of Sun Yat-sen University, ; Guangzhou, 510060 China
                [5 ]ISNI 0000 0001 2360 039X, GRID grid.12981.33, Center for Translational Medicine, The First Affiliated Hospital, , Sun Yat-sen University, ; Guangzhou, 510080 China
                [6 ]ISNI 0000 0001 2360 039X, GRID grid.12981.33, Department of Biochemistry and Molecular Biology, Zhongshan School of Medicine, , Sun Yat-Sen University, ; Guangzhou, 510080 China
                [7 ]ISNI 0000 0000 9889 6335, GRID grid.413106.1, State Key Laboratory of Molecular Oncology, , Chinese Academy of Medical Science and Peking Union Medical College, ; Beijing, 100021 China
                Author information
                http://orcid.org/0000-0001-9771-8534
                Article
                1038
                10.1186/s12943-019-1038-7
                6589893
                31230592
                9c0a2eee-cc77-4ca1-be10-2e6a53c2a287
                © The Author(s). 2019

                Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                History
                : 28 March 2019
                : 19 June 2019
                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/501100001809, National Natural Science Foundation of China;
                Award ID: 81871951
                Award ID: 81772614
                Categories
                Research
                Custom metadata
                © The Author(s) 2019

                Oncology & Radiotherapy
                colorectal cancer,n6-methyladenosine (m6a),mettl3,sox2,igf2bp2
                Oncology & Radiotherapy
                colorectal cancer, n6-methyladenosine (m6a), mettl3, sox2, igf2bp2

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