Abstract
Although water buffaloes are the main milk-producing animals in Indian subcontinent, only limited attempts have been made to identify canonical pathways and gene regulatory networks operating within the mammary glands of these animals. Such information is important for identifying unique transcriptome signatures in the mammary glands of diseased animals. In this report, we analyzed the transcription profile of 3 prepubertal buffalo mammary glands and identified common genes (mean FPKM > 0.2 in all samples) operating in the glands. Among 19,994 protein coding genes, 14,678 genes expressed and 5316 unique genes did not express in prepubertal buffalo mammary glands. Of these 14,678 expressed genes, 79% comprised a ubiquitous transcriptome that was dominated by very lowly expressed genes (51%). The percentage of rarely, moderately, and abundantly expressed genes was 25%, 2%, and 1%, respectively. Gene Ontology (GO) terms reflected in the expression of common genes (mean FPKM > 5.0) for molecular function were related to binding and catalytic activity. Products of these genes were involved in metabolic and cellular processes and belong to nucleic acid binding proteins. The canonical pathways for growth of mammary glands included integrin signaling, inflammation, GnRH and Wnt pathways. KEGG enriched pathways revealed many pathways of cancer including ribosome, splisosome, endocytosis, and ubiquitin-mediated proteolysis, pathways for viral infection, and bacterial invasion of epithelial. Highly expressed genes (mean FPKM > 500 included beta-actin (ACTB), beta-2 microglobulin (B2M), caseins (CSN2, CNS3), collagens (COL1A1, COL3A1), translation elongation factors (EEF1A1, EEF1G, EEF2), keratins (KRT15, KRT19), major histocompatibility complex genes (CD74, JSP.1), vimentin (VIM), and osteopontin (SPP1). Interestingly, expression of milk protein genes in prepubertal glands opens possible roles of these genes in development of mammary glands. We report the whole transcriptomic signature of prepubertal buffalo mammary gland and indicated its molecular signature is similar to cancer type.
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Acosta D, Bagchi S, Broin PÓ, Hollern D, Racedo SE, Morrow B, Sellers RS, Greally JM, Golden A, Andrechek E, Wood T, Montagna C (2016) LPA receptor activity is basal specific and coincident with early pregnancy and involution during mammary gland postnatal development. Sci Rep 6:35810. https://doi.org/10.1038/srep35810
Baldus SE, Wienand JR, Werner JP et al (2005) Expression of MUC1, MUC2 and oligosaccharide epitopes in breast cancer: prognostic significance of a sialylated MUC1 epitope. Int J Oncol 27:1289–1297
Baldwin RL, Wu S, Li W et al (2012) Quantification of transcriptome responses of the rumen epithelium to butyrate infusion using RNA-seq technology. Gene Regul Syst Biol 6:67–80. https://doi.org/10.4137/GRSB.S9687
Cai C, Rajaram M, Zhou X, Liu Q, Marchica J, Li J, Powers RS (2012) Activation of multiple cancer pathways and tumor maintenance function of the 3q amplified oncogene FNDC3B. Cell Cycle 11:1773–1781. https://doi.org/10.4161/cc.20121
Capuco AV, Ellis SE (2013) Comparative aspects of mammary gland development and homeostasis. Annu Rev Anim Biosci 1:179–202. https://doi.org/10.1146/annurev-animal-031412-103632
Chalick M, Jacobi O, Pichinuk E, Garbar C, Bensussan A, Meeker A, Ziv R, Zehavi T, Smorodinsky NI, Hilkens J, Hanisch FG, Rubinstein DB, Wreschner DH (2016) MUC1-ARF—a novel MUC1 protein that resides in the nucleus and is expressed by alternate reading frame translation of MUC1 mRNA. PLoS One 11:e0165031. https://doi.org/10.1371/journal.pone.0165031
Cheng S, Mao Q, Dong Y, Ren J, Su L, Liu J, Liu Q, Zhou J, Ye X, Zheng S, Zhu N (2017) GNB2L1 and its O-GlcNAcylation regulates metastasis via modulating epithelial-mesenchymal transition in the chemoresistance of gastric cancer. PLoS One 12:e0182696. https://doi.org/10.1371/journal.pone.0182696
Choudhary RK, Pathak D, Deka D et al (2013) Vimentin identifies myoepithelial cells of buffalo mammary tissue. Rum Sci 2:157–162
Choudhary RK, Pathak D, Deka D, Ramneek (2014a) Cellular composition of water buffalo mammary gland and its proliferation status during dry and mastitis (Abstract only). J Dairy Sci 97(E-Suppl 1):207
Choudhary RK, Pathak D, Dipak D et al (2014b) Vimentin identifies myoepithelial cells of buffalo mammary tissue. Rum Sci 2:157–161
Choudhary RK, Choudhary S, Kaur H, Pathak D (2016) Expression of putative stem cell marker, hepatocyte nuclear factor 4 alpha, in mammary gland of water buffalo. Anim Biotechnol 27:182–189. https://doi.org/10.1080/10495398.2016.1164179
Choudhary RK, Choudhary S, Verma R (2018) In vivo response of xanthosine on mammary gene expression of lactating Beetal goat. Mol Biol Rep in press. https://doi.org/10.1007/s11033-018-4196-6
Connor EE, Wood DL, Sonstegard TS et al (2005) Chromosomal mapping and quantitative analysis of estrogen-related receptor alpha-1, estrogen receptors alpha and beta and progesterone receptor in the bovine mammary gland. J Endocrinol 185:593–603. https://doi.org/10.1677/joe.1.06139
Crisà A, Ferrè F, Chillemi G, Moioli B (2016) RNA-sequencing for profiling goat milk transcriptome in colostrum and mature milk. BMC Vet Res 12:264. https://doi.org/10.1186/s12917-016-0881-7
Cui X, Hou Y, Yang S, Xie Y, Zhang S, Zhang Y, Zhang Q, Lu X, Liu GE, Sun D (2014) Transcriptional profiling of mammary gland in Holstein cows with extremely different milk protein and fat percentage using RNA sequencing. BMC Genomics 15:226. https://doi.org/10.1186/1471-2164-15-226
Dai WT, Zou YX, White RR et al (2018) Transcriptomic profiles of the bovine mammary gland during lactation and the dry period. Funct Integr Genomics 18:125–140. https://doi.org/10.1007/s10142-017-0580-x
Friedenson B (2013) Mutations in components of antiviral or microbial defense as a basis for breast cancer. Funct Integr Genomics 13:411–424. https://doi.org/10.1007/s10142-013-0336-1
Gao Y, Lin X, Shi K, Yan Z, Wang Z (2013) Bovine mammary gene expression profiling during the onset of lactation. PLoS One 8:e70393. https://doi.org/10.1371/journal.pone.0070393
Ginestier C, Hur MH, Charafe-Jauffret E, Monville F, Dutcher J, Brown M, Jacquemier J, Viens P, Kleer CG, Liu S, Schott A, Hayes D, Birnbaum D, Wicha MS, Dontu G (2007) ALDH1 is a marker of normal and malignant human mammary stem cells and a predictor of poor clinical outcome. Cell Stem Cell 1:555–567. https://doi.org/10.1016/j.stem.2007.08.014
Goudarzi KM, Lindström MS (2016) Role of ribosomal protein mutations in tumor development (review). Int J Oncol 48:1313–1324. https://doi.org/10.3892/ijo.2016.3387
Hassan MK, Kumar D, Naik M, Dixit M (2018) The expression profile and prognostic significance of eukaryotic translation elongation factors in different cancers. PLoS One 13:e0191377. https://doi.org/10.1371/journal.pone.0191377
Huang DW, Sherman BT, Lempicki RA (2009) Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc 4:44–57. https://doi.org/10.1038/nprot.2008.211
Janjanam J, Singh S, Jena MK, Varshney N, Kola S, Kumar S, Kaushik JK, Grover S, Dang AK, Mukesh M, Prakash BS, Mohanty AK (2014) Comparative 2D-DIGE proteomic analysis of bovine mammary epithelial cells during lactation reveals protein signatures for lactation persistency and milk yield. PLoS One 9:e102515. https://doi.org/10.1371/journal.pone.0102515
Jena MK, Janjanam J, Naru J, Kumar S, Kumar S, Singh S, Mohapatra SK, Kola S, Anand V, Jaswal S, Verma AK, Malakar D, Dang AK, Kaushik JK, Reddy VS, Mohanty AK (2015) DIGE based proteome analysis of mammary gland tissue in water buffalo (Bubalus bubalis): lactating vis-a-vis heifer. J Proteome 119:100–111. https://doi.org/10.1016/j.jprot.2015.01.018
Kapila N, Kishore A, Sodhi M, Sharma A, Kumar P, Mohanty AK, Jerath T, Mukesh M (2013) Identification of appropriate reference genes for qRT-PCR analysis of heat-stressed mammary epithelial cells in riverine buffaloes (Bubalus bubalis). ISRN Biotechnol 2013:1–9. https://doi.org/10.5402/2013/735053
Koval A, Katanaev VL (2018) Dramatic dysbalancing of the Wnt pathway in breast cancers. Sci Rep 8:7329. https://doi.org/10.1038/s41598-018-25672-6
Kukurba KR, Montgomery SB (2015) RNA sequencing and analysis. Cold Spring Harb Protoc 2015:951–969. https://doi.org/10.1101/pdb.top084970
Li RW, Schroeder SG (2011) Cytoskeleton remodeling and alterations in smooth muscle contractility in the bovine jejunum during nematode infection. Funct Integr Genomics. https://doi.org/10.1007/s10142-011-0259-7
Li J-J, Xie D (2015) RACK1, a versatile hub in cancer. Oncogene 34:1890–1898. https://doi.org/10.1038/onc.2014.127
Lim E, Wu D, Pal B, Bouras T, Asselin-Labat ML, Vaillant F, Yagita H, Lindeman GJ, Smyth GK, Visvader JE (2010) Transcriptome analyses of mouse and human mammary cell subpopulations reveal multiple conserved genes and pathways. Breast Cancer Res 12:R21. https://doi.org/10.1186/bcr2560
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25:402–408. https://doi.org/10.1006/meth.2001.1262
Lv Q-L, Huang Y-T, Wang G-H, Liu YL, Huang J, Qu Q, Sun B, Hu L, Cheng L, Chen SH, Zhou HH (2016) Overexpression of RACK1 promotes metastasis by enhancing epithelial-mesenchyal transition and predicts poor prognosis in human glioma. Int J Environ Res Public Health 13:1021. https://doi.org/10.3390/ijerph13101021
Mani SA, Guo W, Liao M et al (2009) The epithelial-mesenchymal transition generates cells with properties of stem cells. Cell 133:704–715. https://doi.org/10.1016/j.cell.2008.03.027.The
Mi H, Huang X, Muruganujan A, Tang H, Mills C, Kang D, Thomas PD (2017) PANTHER version 11: expanded annotation data from Gene Ontology and Reactome pathways, and data analysis tool enhancements. Nucleic Acids Res 45:D183–D189. https://doi.org/10.1093/nar/gkw1138
Nath S, Mukherjee P (2014) MUC1: a multifaceted oncoprotein with a key role in cancer progression. Trends Mol Med 20:332–342. https://doi.org/10.1016/j.molmed.2014.02.007
Oji Y, Tatsumi N, Fukuda M et al (2014) The translation elongation factor eEF2 is a novel tumor-associated antigen overexpressed in various types of cancers. Int J Oncol 44:1461–1469. https://doi.org/10.3892/ijo.2014.2318
Osińska E, Wicik Z, Godlewski MM, Pawłowski K, Majewska A, Mucha J, Gajewska M, Motyl T (2014) Comparison of stem/progenitor cell number and transcriptomic profile in the mammary tissue of dairy and beef breed heifers. J Appl Genet 55:383–395. https://doi.org/10.1007/s13353-014-0213-1
Pohl S-G, Brook N, Agostino M, Arfuso F, Kumar AP, Dharmarajan A (2017) Wnt signaling in triple-negative breast cancer. Oncogenesis 6:e310–e310. https://doi.org/10.1038/oncsis.2017.14
Rahn JJ, Dabbagh L, Pasdar M, Hugh JC (2001) The importance of MUC1 cellular localization in patients with breast carcinoma. Cancer 91:1973–1982. https://doi.org/10.1002/1097-0142(20010601)91:11<1973::AID-CNCR1222>3.0.CO;2-A
Rittling S, Novick K (1997) Osteopontin expression in mammary gland development and tumorigenesis. Cell Growth Differ 8:1061–1069
Rodrigues LR, Teixeira JA, Schmitt FL, Paulsson M, Lindmark-Mansson H (2007) The role of osteopontin in tumor progression and metastasis in breast cancer. Cancer Epidemiol Biomark Prev 16:1087–1097. https://doi.org/10.1158/1055-9965.EPI-06-1008
Saleh S, Thompson DE, McConkey J, Murray P, Moorehead RA (2016) Osteopontin regulates proliferation, apoptosis, and migration of murine claudin-low mammary tumor cells. BMC Cancer 16:359. https://doi.org/10.1186/s12885-016-2396-9
Schmittgen TD, Livak KJ (2008) Analyzing real-time PCR data by the comparative CT method. Nat Protoc 3:1101–1108. https://doi.org/10.1038/nprot.2008.73
Singh M, Thomson PC, Sheehy PA, Raadsma HW (2013) Comparative transcriptome analyses reveal conserved and distinct mechanisms in ovine and bovine lactation. Funct Integr Genomics 13:115–131. https://doi.org/10.1007/s10142-012-0307-y
Stein T, Salomonis N, Nuyten DSA, van de Vijver MJ, Gusterson BA (2009) A mouse mammary gland involution mRNA signature identifies biological pathways potentially associated with breast cancer metastasis. J Mammary Gland Biol Neoplasia 14:99–116. https://doi.org/10.1007/s10911-009-9120-1
Stires H, Crismale-Gann C, Belden WJ, Cohick WS (2016) The prepubertal mammary gland transcriptome suggests a role for the immune system in hormone-independent breast cancer. Cancer Res 76:785–785. https://doi.org/10.1158/1538-7445.AM2016-785
Suárez-Vega A, Gutiérrez-Gil B, Klopp C, Tosser-Klopp G, Arranz JJ (2016) Comprehensive RNA-Seq profiling to evaluate lactating sheep mammary gland transcriptome. Sci Data 3:160051. https://doi.org/10.1038/sdata.2016.51
Szklarczyk D, Franceschini A, Wyder S, Forslund K, Heller D, Huerta-Cepas J, Simonovic M, Roth A, Santos A, Tsafou KP, Kuhn M, Bork P, Jensen LJ, von Mering C (2015) STRING v10: protein-protein interaction networks, integrated over the tree of life. Nucleic Acids Res 43:D447–D452. https://doi.org/10.1093/nar/gku1003
Trapnell C, Pachter L, Salzberg SL (2009) TopHat: discovering splice junctions with RNA-Seq. Bioinformatics 25:1105–1111. https://doi.org/10.1093/bioinformatics/btp120
Wang M-H, Sun R, Zhou X-M, Zhang MY, Lu JB, Yang Y, Zeng LS, Yang XZ, Shi L, Xiao RW, Wang HY, Mai SJ (2018) Epithelial cell adhesion molecule overexpression regulates epithelial-mesenchymal transition, stemness and metastasis of nasopharyngeal carcinoma cells via the PTEN/AKT/mTOR pathway. Cell Death Dis 9:2. https://doi.org/10.1038/s41419-017-0013-8
Wickramasinghe S, Rincon G, Islas-Trejo A, Medrano JF (2012) Transcriptional profiling of bovine milk using RNA sequencing. BMC Genomics 13:45. https://doi.org/10.1186/1471-2164-13-45
Zhang X, Liu N, Ma D, Liu L, Jiang L, Zhou Y, Zeng X, Li J, Chen Q (2016) Receptor for activated C kinase 1 (RACK1) promotes the progression of OSCC via the AKT/mTOR pathway. Int J Oncol 49:539–548. https://doi.org/10.3892/ijo.2016.3562
Acknowledgements
The authors also would like to thank Drs. Anthony Capuco and Kristy Daniels for scientific discussion and Dr. Capuco for grammatical usage and language correction. The authors would also like to thank Dr. Kuldeep Gupta, Professor of Veterinary Pathology, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana in identifying pathology of these mammary tissues.
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ESM 1
Ubiquitous gene list and their mean FPKM values (>0.2 in all three samples), identified in prepubertal buffalo mammary tissue (XLSX 5093 kb)
ESM 2
Enrichment of functional annotations using Gene Ontology (GO) terms for Biological Process, Molecular Function and Cellular Component of ubiquitous genes (XLSX 99 kb)
ESM 3
Significant KEGG pathways of all the ubiquitous genes having mean FPKM >5.0 (XLSX 32 kb)
ESM 4
Pathways that were not expressed in prepubertal buffalo mammary gland. (XLSX 14 kb)
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Choudhary, R.K., Choudhary, S., Mukhopadhyay, C.S. et al. Deciphering the transcriptome of prepubertal buffalo mammary glands using RNA sequencing. Funct Integr Genomics 19, 349–362 (2019). https://doi.org/10.1007/s10142-018-0645-5
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DOI: https://doi.org/10.1007/s10142-018-0645-5