Genome-wide DNA methylome and whole-transcriptome landscapes of spontaneous intraductal papilloma in tree shrews.

Original Article Page 1 of 19 Genome-wide DNA methylome and whole-transcriptome landscapes of spontaneous intraductal papilloma in tree shrews Chengxiu Liu, Yuanyuan Han, Pinfen Tong, Dexuan Kuang, Na Li, Caixia Lu, Xiaomei Sun, Wenguang Wang, Jiejie Dai The Center of Tree Shrew Germplasm Resources, Institute of Medical Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, Kunming, China Contributions: (I) Conception and design: J Dai, C Liu; (II) Administrative support: None; (III) Provision of study materials or patients: C Liu, Y Han; (IV) Collection and assembly of data: D Kuang, C Liu, N Li, W Wang, P Tong; (V) Data analysis and interpretation: X Sun, J Dai; (VI) Manuscript writing: All authors; (VII) Final approval of manuscript: All authors. Correspondence to: Jiejie Dai. Center of Tree Shrew Germplasm Resources, Institute of Medical Biology, Chinese Academy of Medical Sciences/Peking Union Medical College, Kunming 650118, China. Email: . Background: Breast intraductal papilloma (IP) is mainly caused by the abnormal proliferation of ductal epithelial cells. Tree shrews have potential as an animal model for the study of breast tumours; however, little is known regarding the transcriptome and DNA methylome landscapes of breast IP in tree shrews. In this research, we conducted whole-genome DNA methylation and transcriptome analyses of breast IP and normal mammary glands in tree shrews. Methods: DNA methylation profiles were generated from the whole-genome bisulfite sequencing and whole-transcriptome landscapes of IP and control groups of tree shrews through strand-specific library construction and RNA sequencing. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes functional enrichment analyses and gene set enrichment analysis were performed. Spearman’s correlation analysis was used to identify statistical relationships between gene expression and DNA methylation. Results: A genome-wide perspective of the epigenetic regulation of protein-coding genes in breast IP in tree shrews was obtained. The methylation levels at CG sites were considerably higher than those at CHG or CHH sites, and were highest in gene body regions. In total, 3,486, 82 and 361 differentially methylated regions (DMRs) were identified in the context of CG, CHG, and CHH, respectively, and 701 differentially methylated genes (DMGs) were found. Further, through transcriptomic analysis, 62 differentially expressed genes, 50 long noncoding RNAs, and 32 circular RNAs were identified in breast IP compared to normal mammary glands. Correlation analysis between the DNA methylation and transcriptome data revealed that 25 DMGs were also differentially expressed genes, among which the expression levels of 9 genes were negatively correlated with methylation levels in gene body regions. Importantly, integrated analysis identified 3 genes (PDZ domain-containing 1, ATPase plasma membrane Ca2+ transporting 4 and Lymphocyte cytosolic protein 1) that could serve as candidates for further study of breast IP in tree shrews. Conclusions: This research has unearthed the comprehensive landscape of the transcriptome and DNA methylome of spontaneous IP in tree shrews, as well as candidate tumorigenesis related genes in IP. These results will contribute to the use of tree shrews in animal models of breast tumours. Keywords: Tree shrew; spontaneous breast intraductal papilloma (spontaneous breast IP); DNA methylation; transcriptome Submitted Feb 04, 2021. Accepted for publication Apr 17, 2021. doi: 10.21037/atm-21-1293 View this article at: http://dx.doi.org/10.21037/atm-21-1293 © Annals of Translational Medicine. All rights reserved. Ann Transl Med 2021;9(8):688 | http://dx.doi.org/10.21037/atm-21-1293 Page 2 of 19 Liu et al. DNA methylome and transcriptome of breast IP in tree shrews Introduction Intraductal papilloma (IP), a benign tumour that forms in the breast ducts, accounts for approximately 10% of cases of benign breast lesions (1). IP, which is caused by the abnormal proliferation of ductal epithelial cells, most commonly affects women between the ages of 35 and 55 years old (2). Hormones, fertility, and diet are all risk factors that predispose women to the development of IP (3). Because IP is related to atypia, ductal carcinoma in situ (DCIS), and carcinoma, it is classified as a high-risk precursor lesion and carries a 6.3% risk of malignancy (3); upon surgical excision, IP may be upgraded to atypical ductal hyperplasia or DCIS (4). However, at present, the mechanism of breast neoplasms is not fully understood, and multidimensional molecular data from IP patients have not been fully integrated in studies on this topic. The results of DNA sequencing research have confirmed that tree shrews are closely related to primates (5). Consequently, tree shrews have become an increasingly popular experimental animal model for various human tumours, including lung cancer (6), hepatocellular carcinoma (7), and glioblastoma (8). Genome sequencing of Chinese tree shrews was first accomplished in 2013 and has provided a useful resource for functional genomic studies since (9). A database of the genome sequencing data of tree shrews has also been established (10). Most importantly, in terms of morphology and structure, the mammary glands of tree shrews are similar to those of humans (11). Based on these qualities, tree shrews are ideal experimental animals for studying the pathogenesis of mammary tumours. However, few studies have used tree shrews as a novel breast tumour animal model to examine gene expression patterns and the underlying function of DNA methylation in the tumorigenesis of spontaneous IP. DNA methylation is one of the epigenetic changes that has been shown to play a key role in the pretranscriptional regulation and inhibition of gene expression in multiple mammalian genomes. The mapping of genome-wide DNA methylation is of great importance to understanding tumorigenesis (12). DNA methylation is implicated in many cancers, including thyroid cancer (13), non-small cell lung cancer (14), and gastric cancer (15), as well as in the development and progression of breast cancer (16). Limited evidence has also indicated that the aberrant methylation of cytosine residues is involved in the development of IP (17). Therefore, delineating the DNA methylation profile and identifying differentially methylated genes (DMGs) in IP © Annals of Translational Medicine. All rights reserved. would be helpful to understanding the tumorigenesis of papilloma from the perspective of epigenetic regulation. It is generally believed that the abnormal reprogramming of the whole transcriptome, including genes, long noncoding RNAs (lncRNAs), and circular RNAs (circRNAs), is a crucial process in the occurrence and progression of tumours. Recently, RNA sequencing (RNA-seq) studies of breast cancer (18) have been conducted to inform a deeper understanding of the mechanisms involved, and research on the (...truncated)