ORIGINAL RESEARCH published: 26 January 2021 doi: 10.3389/fcell.2020.629030 Dynamic Expression of m6A Regulators During Multiple Human Tissue Development and Cancers Ya Zhang 1,2†, Sicong Xu 1,2†, Gang Xu 1,2, Yueying Gao 1,2, Si Li 1,2, Ke Zhang 1,2, Zhanyu Tian 1,2, Jing Guo 1,2, Xia Li 1,2*, Juan Xu 1,2* and Yongsheng Li 1,2* 1 Key Laboratory of Tropical Translational Medicine of Ministry of Education, Hainan Medical University, Haikou, China, 2 College of Biomedical Information and Engineering, Hainan Medical University, Haikou, China N6-methyladenosine (m6A) plays critical roles in human development and cancer Edited by: progression. However, our knowledge regarding the dynamic expression of m6A Meng Zhou, Wenzhou Medical University, China regulators during human tissue development is still lacking. Here, we comprehensively 6 Reviewed by: analyzed the dynamic expression alterations of m A regulators during seven tissue Jian Huang, development and eight cancer types. We found that m6A regulators globally exhibited University of Electronic Science and Technology of China, China decreased expression during development. In addition, IGF2BP1/2/3 (insulinlike growth Lixin Cheng, factor 2 MRNA-binding protein 1/2/3) exhibited reverse expression pattern in cancer Jinan University, China progression, suggesting an oncofetal reprogramming in cancer. The expressions Kuixi Zhu, University of Arizona, United States of IGF2BP1/2/3 were regulated by genome alterations, particularly copy number *Correspondence: amplification in cancer. Clinical association analysis revealed that higher expressions of Yongsheng Li IGF2BP1/2/3 were associated with worse survival of cancer patients. Finally, we found [email protected] Juan Xu that genes significantly correlated with IGF2BP1/2/3 were significantly enriched in cancer [email protected] hallmark-related pathways. In summary, dynamic expression analysis will guide both Xia Li mechanistic and therapeutic roles of m6A regulators during tissue development and [email protected] cancer progression. †These authors have contributed 6 equally to this work Keywords: m A regulators, tissue development, cancer, gene experession, survival Specialty section: This article was submitted to INTRODUCTION Epigenomics and Epigenetics, 6 a section of the journal N6-methyladenosine (m A) is the most abundant RNA modification and has been shown to play Frontiers in Cell and Developmental important roles in development and cancer (Dominissini et al., 2012; Meyer et al., 2012). RNA Biology methylation has been shown to be catalyzed by a multicomponent methyltransferase complex, Received: 13 November 2020 including METTL13/14 and WTAP (Liu et al., 2014). RNA methylation has been found in Accepted: 21 December 2020 thousands of coding and non-coding genes. However, we still lack knowledge about the functions Published: 26 January 2021 of m6A regulators in development and cancer. Citation: Numerous studies have shown that m6A regulators were widely perturbed in various types Zhang Y, Xu S, Xu G, Gao Y, Li S, of cancer. METTL3 and IGF2BP2 (insulinlike growth factor 2 MRNA-binding protein 2) were Zhang K, Tian Z, Guo J, Li X, Xu J and overexpressed in colorectal cancer and promote the cancer progression (Li et al., 2019). WTAP was Li Y (2021) Dynamic Expression of 6 also shown to lead to suppression of ETS1 and contribute to the proliferation of liver cancer (Chen m A Regulators During Multiple 6 Human Tissue Development and et al., 2019b). Xu et al. found that 19 m A regulators were highly expressed in glioma tissues, and Cancers. the expression of regulators was associated with prognoses and grade (Xu et al., 2020b). One recent Front. Cell Dev. Biol. 8:629030. study has revealed an essential role of YTHDF1 in the progression of hepatocellular carcinoma doi: 10.3389/fcell.2020.629030 (Liu et al., 2020). RNA methyltransferase METTL3 has been found to facilitate colorectal cancer by Frontiers in Cell and Developmental Biology | www.frontiersin.org 1 January 2021 | Volume 8 | Article 629030 Zhang et al. m6A Regulators in Development and Cancer activating m6A-GLUT1-mTORC1 axis and is a therapeutic preprocessed with RMA normalization, merged, and batch target (Chen et al., 2020). Moreover, we recently performed effect–corrected via Combat method (Leek et al., 2012). comprehensive characterization of m6A regulators in 33 cancer types and found that there were widespread expression 6 6 Collection of m A Regulators perturbation of m A regulators in cancer (Li et al., 2019a). The gene list of m6A regulators was obtained from one of our However, there has been no research that comprehensively 6 studies (Li et al., 2019b), which curated a catalog of 20 genes that explored the expression landscape of m A regulators during function mainly as regulators of RNA methylation. In total, there human tissue development. were 11 readers, 7 writers, and 2 erasers. All these gene symbols In this study, we performed a comprehensive evaluation were converted into Ensemble gene IDs and HGNC symbols of the expression of m6A regulators across multiple human 6 based on annotation from GeneCards (https://www.genecards. tissues development and cancer. We found that the m A org/). regulators exhibited decreased expression after born. Moreover, m6A regulators were up-regulated in cancer, which suggests an oncofetal reprogramming mechanism in cancer. Moreover, Differential Expression Analysis 6 we investigated the association between the expression of m6A To evaluate whether the expression of m A regulators exhibits regulators and patient survival. Our comprehensive analysis differential expression between normal and cancer, we used the of the dynamic expression of m6A regulators provided novel Wilcoxon rank sum test to compare the expression levels. Here, insights into their function in development and cancer. only cancer types with more than five normal samples were analyzed. The gene expression of m6A regulators across normal or cancer samples was shown by box plots. MATERIALS AND METHODS Transcriptome During Human Tissue Genome Alteration of m6A Regulators 6 Development To explore the genome alteration of m A regulators, we used the cBioPortal tool (Gao et al., 2013), which provided a Web resource Gene expression data across human tissue development were for exploring, visualizing, and analyzing multidimensional downloaded from ArrayExpress with the accession code E- cancer genomic data. The gene symbols of m6A regulators were MTAB-6814 (Cardoso-Moreira et al., 2019). The samples were used as input, and TCGA pan-cancer datasets were used. The started from prenatal development at 4 weeks postconception genome alteration frequency of m6A regulators was calculated (WPC) to 20 WPC. Moreover, postnatal samples were also and plotted as bar plot. Here, genetic mutations and copy number sampled, including infants, toddlers, school, teenagers, and adults variation were considered. from each decade until 65 years of age. There were seven human tissues analyzed, including brain, cerebellum, heart, kidney, liver, ovary, and testis. In total, 297 human samples were sequenced Survival Analysis by RNA-Seq. Gene expression levels were calculated as reads per To explore whether the expression of m6A regulators was kilobase of exon model per million mapped reads. associated with patient survival, we divided all the patients into two groups based on the median expression of each m6A Gene Expression in Human Cancers regulator. The log-rank test was used to test the difference We downloaded the gene expression across human cancer survival rates between two groups. This process was performed by the survival package in R program (https://cran.r-project.org/ types from The Cancer Genome Atlas (TCGA) project (Cancer < Genome Atlas Research et al., 2013). According to the tissues in web/packages/survival/index.html). The p 0.05 was considered human development, we considered eight cancer types, including as significant. The odds ratios and 95% confidence levels were kidney renal clear cell carcinoma (KIRC), kidney renal papillary also calculated and shown by ggplot2 package in R program cell carcinoma (KIRP), kidney chromophobe (KICH), brain (Wickham, 2009). lower grade glioma, glioblastoma multiforme, ovarian serous cystadenocarcinoma, liver hepatocellular carcinoma (LIHC), Function Prediction of Regulators and testicular germ cell tumors. Gene expression levels were To predict the function of m6A regulators in human development calculated as fragments per kilobase of exon model per million and cancer, we first calculated the Spearman correlation mapped fragments. Moreover, we downloaded the clinical coefficient (SPCC) between the expression of m6A regulators information of patients, including the survival time, survival and other protein coding genes. All the genes were ranked by status from TCGA project. SPCC and subjected to preranked Gene Set Enrichment Analysis To validate the expression of IGF2BPs regulators across cancer (GSEA) (Mootha et al., 2003; Subramanian et al., 2005). The types, we collected gene expression data across ∼7,400 samples cancer hallmark-related functions were considered (Liberzon representing 11 cancers. Gene expression data were collected et al., 2011, 2015). Gene sets with more than 5 genes and fewer from Gene Expression Omnibus. To minimize interplatform than 1,500 genes were considered. We performed 1,000 times variation, only datasets generated from the Affymetrix Human randomization. The normalized enrichment scores of GSEA were