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Comparative RNA-seq analysis and ceRNA network of genistein-treated GT1-7 neurons

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Abstract

Background

Genistein is an isoflavone and phytoestrogen originated from soybean and soy products. Due to the close structural and functional proximity towards 17β-estradiol, genistein has been suggested to influence endocrine and reproductive systems. Previous studies showed that genistein could affect hypothalamic–pituitary–gonadal (HPG) axis and impact gonadotropin-releasing hormone (GnRH) secretion in hypothalamic GT1-7 neurons. However, the underlying mechanism remains mostly unknown.

Objectives

Comparative transcriptomic analyses of mRNAs, long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) were performed in genistein-treated GT1-7 cells by high-throughput RNA sequencing. Competing endogenous RNA (ceRNA) networks were constructed based on potential interactions in lncRNAs, miRNAs and mRNAs.

Results

Compared to the control, 1134, 1126 and 30 differentially expressed mRNA, lncRNA and miRNAs were identified. The most significantly upregulated mRNA was growth-regulating estrogen receptor binding 1 (Greb1), possibly related to the increased levels of estrogen receptors (Esr1 and Esr2). Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses demonstrated that genistein interfered with cell cycle, metabolic processes, as well as GnRH and mitogen-activated protein kinase signaling pathways in GT1-7 cells. CeRNA networks predicted that prostatic cancer-related miRNA mmu-miR-212-5p and its targeted genes Phf2 and Aldh3b1 might be associated with the regulation of genistein-induced GnRH secretion in GT1-7 cells, and 27 lncRNAs could completely interact with mmu-miR-212-5p and downregulate the transcription of target genes.

Conclusion

Results from the study could provide potential targets of both mRNA and non-coding RNAs for further studies to explore the endocrine-interfering effects of genistein.

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References

  • Al-Kawlani B et al (2020) Doxorubicin induces cytotoxicity and miR-132 expression in granulosa cells. Reprod Toxicol 96:95–101

    Article  CAS  PubMed  Google Scholar 

  • Arispe SA, Adams B, Adams TE (2013) Effect of phytoestrogens on basal and GnRH-induced gonadotropin secretion. J Endocrinol 219:243–250

    Article  CAS  PubMed  Google Scholar 

  • Bateman HL, Patisaul HB (2008) Disrupted female reproductive physiology following neonatal exposure to phytoestrogens or estrogen specific ligands is associated with decreased GnRH activation and kisspeptin fiber density in the hypothalamus. Neurotoxicology 29:988–997

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Bliss SP, Navratil AM, Xie J, Roberson MS (2010) GnRH signaling, the gonadotrope and endocrine control of fertility. Front Neuroendocrinol 31(3):322–340. https://doi.org/10.1016/j.yfrne.2010.04.002

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Cao C et al (2018) Reproductive role of miRNA in the hypothalamic-pituitary axis. Mol Cell Neurosci 88:130–137

    Article  CAS  PubMed  Google Scholar 

  • Chen H et al (2019) Identification and functional characterization of microRNAs in rat Leydig cells during development from the progenitor to the adult stage. Mol Cell Endocrinol 493:110453

    Article  CAS  PubMed  Google Scholar 

  • Cheng W, Li XW, Xiao YQ, Duan SB (2019) Non-coding RNA-associated ceRNA networks in a new contrast-induced acute kidney injury rat model. Mol Ther Nucleic Acids 17:102–112

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Day FR et al (2017) Genomic analyses identify hundreds of variants associated with age at menarche and support a role for puberty timing in cancer risk. Nat Genet 49:834–841

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Dey BK, Mueller AC, Dutta A (2014) Long non-coding RNAs as emerging regulators of differentiation, development, and disease. Transcription 5:e944014

    Article  PubMed  PubMed Central  Google Scholar 

  • D’Occhio MJ, Baruselli PS, Campanile G (2019) Influence of nutrition, body condition, and metabolic status on reproduction in female beef cattle: a review. Theriogenology 125:277–284

    Article  CAS  PubMed  Google Scholar 

  • Fan YX et al (2018) Effect of dietary energy restriction and subsequent compensatory feeding on testicular transcriptome in developing rams. Theriogenology 119:198–207

    Article  CAS  PubMed  Google Scholar 

  • Fuqua JS (2013) Treatment and outcomes of precocious puberty: an update. J Clin Endocrinol Metab 98:2198–2207

    Article  CAS  PubMed  Google Scholar 

  • Guzmán A, Hernández-Coronado CG, Rosales-Torres AM, Hernández-Medrano JH (2019) Leptin regulates neuropeptides associated with food intake and GnRH secretion. Ann Endocrinol (paris) 80:38–46

    Article  PubMed  Google Scholar 

  • Hayder H et al (2021) Overexpression of miR-210-3p impairs extravillous trophoblast functions associated with uterine spiral artery remodeling. Int J Mol Sci 22:3961

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Jaiswal N, Akhtar J, Singh SP, Badruddeen AF (2019) An overview on genistein and its various formulations. Drug Res (stuttg) 69:305–313

    Article  CAS  PubMed  Google Scholar 

  • Kanasaki H, Oride A, Mijiddorj T, Sukhbaatar U, Kyo S (2017) How is GnRH regulated in GnRH-producing neurons? Studies using GT1-7 cells as a GnRH-producing cell model. Gen Comp Endocrinol 247:138–142

    Article  CAS  PubMed  Google Scholar 

  • Kim KH et al (2020) Human placenta-derived mesenchymal stem cells stimulate ovarian function via miR-145 and bone morphogenetic protein signaling in aged rats. Stem Cell Res Ther 11:472

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kuiper GG et al (1998) Interaction of estrogenic chemicals and phytoestrogens with estrogen receptor beta. Endocrinology 139:4252–4263

    Article  CAS  PubMed  Google Scholar 

  • Li Y, Kong D, Ahmad A, Bao B, Sarkar FH (2012) Targeting bone remodeling by isoflavone and 3,3’-diindolylmethane in the context of prostate cancer bone metastasis. PLoS ONE 7:e33011

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Li Y et al (2013) Epigenetic reactivation of estrogen receptor-α (ERα) by genistein enhances hormonal therapy sensitivity in ERα-negative breast cancer. Mol Cancer 12:9

    Article  PubMed  PubMed Central  Google Scholar 

  • Li R, Zhao F, Diao H, Xiao S, Ye X (2014) Postweaning dietary genistein exposure advances puberty without significantly affecting early pregnancy in C57BL/6J female mice. Reprod Toxicol 44:85–92

    Article  CAS  PubMed  Google Scholar 

  • Li D et al (2017) CYP1A1 based on metabolism of xenobiotics by cytochrome P450 regulates chicken male germ cell differentiation. In Vitro Cell Dev Biol Anim 53:293–303

    Article  CAS  PubMed  Google Scholar 

  • Li X et al (2020) Screening and evaluating of long non-coding RNAs in prenatal and postnatal pituitary gland of sheep. Genomics 112:934–942

    Article  CAS  PubMed  Google Scholar 

  • Livadas S, Chrousos GP (2019) Molecular and environmental mechanisms regulating puberty initiation: an integrated approach. Front Endocrinol (lausanne) 10:828

    Article  PubMed  Google Scholar 

  • Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta C(T)) method. Methods 25:402–408

    Article  CAS  PubMed  Google Scholar 

  • Losa SM et al (2011) Neonatal exposure to genistein adversely impacts the ontogeny of hypothalamic kisspeptin signaling pathways and ovarian development in the peripubertal female rat. Reprod Toxicol 31:280–289

    Article  CAS  PubMed  Google Scholar 

  • Love MI, Huber W, Anders S (2014) Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 15:550

    Article  PubMed  PubMed Central  Google Scholar 

  • Machtinger R et al (2017) Extracellular microRNAs in follicular fluid and their potential association with oocyte fertilization and embryo quality: an exploratory study. J Assist Reprod Genet 34:525–533

    Article  PubMed  PubMed Central  Google Scholar 

  • Mahesh VB, Zamorano P, De Sevilla L, Lewis D, Brann DW (1999) Characterization of ionotropic glutamate receptors in rat hypothalamus, pituitary and immortalized gonadotropin-releasing hormone (GnRH) neurons (GT1-7 cells). Neuroendocrinology 69:397–407

    Article  CAS  PubMed  Google Scholar 

  • Mao X, Cai T, Olyarchuk JG, Wei L (2005) Automated genome annotation and pathway identification using the KEGG Orthology (KO) as a controlled vocabulary. Bioinformatics 21:3787–2793

    Article  CAS  PubMed  Google Scholar 

  • Marchitti SA, Brocker C, Orlicky DJ, Vasiliou V (2010) Molecular characterization, expression analysis, and role of ALDH3B1 in the cellular protection against oxidative stress. Free Radic Biol Med 49:1432–1443

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • McGarvey C et al (2001) Phytoestrogens and gonadotropin-releasing hormone pulse generator activity and pituitary luteinizing hormone release in the rat. Endocrinology 142:1202–1208

    Article  CAS  PubMed  Google Scholar 

  • Melamed P et al (2012) Gonadotrophin-releasing hormone signalling downstream of calmodulin. J Neuroendocrinol 24:1463–1475

    Article  CAS  PubMed  Google Scholar 

  • Mercer TR, Mattick JS (2013) Structure and function of long noncoding RNAs in epigenetic regulation. Nat Struct Mol Biol 20:300–207

    Article  CAS  PubMed  Google Scholar 

  • Messina M (2014) Soy foods, isoflavones, and the health of postmenopausal women. Am J Clin Nutr 100(Suppl 1):423S-430S

    Article  CAS  PubMed  Google Scholar 

  • Messina A, Langlet F, Prevot V (2017) MicroRNAs: new players in the hypothalamic control of fertility. Med Sci (paris) 33:506–511

    Article  PubMed  Google Scholar 

  • Mukund V (2020) Genistein: its role in breast cancer growth and metastasis. Curr Drug Metab 21:6–10

    Article  CAS  PubMed  Google Scholar 

  • Pappa S, Padilla N, Iacobucci S, Vicioso M, Álvarez de la Campa E, Navarro C, Marcos E, de la Cruz X, Martínez-Balbás MA (2019) PHF2 histone demethylase prevents DNA damage and genome instability by controlling cell cycle progression of neural progenitors. Proc Natl Acad Sci USA 116(39):19464–19473. https://doi.org/10.1073/pnas.1903188116

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Park C et al (2019) Induction of G2/M cell cycle arrest and apoptosis by genistein in human bladder cancer T24 cells through inhibition of the ROS-dependent PI3k/Akt signal transduction pathway. Antioxidants (basel) 8:327

    Article  CAS  PubMed  Google Scholar 

  • Patisaul HB (2017) Endocrine disruption of vasopressin systems and related behaviors. Front Endocrinol (lausanne) 8:134

    Article  PubMed  Google Scholar 

  • Patisaul HB, Fortino AE, Polston EK (2007) Differential disruption of nuclear volume and neuronal phenotype in the preoptic area by neonatal exposure to genistein and bisphenol-A. Neurotoxicology 28:1–12

    Article  CAS  PubMed  Google Scholar 

  • Qi JC et al (2021) CDK13 upregulation-induced formation of the positive feedback loop among circCDK13, miR-212-5p/miR-449a and E2F5 contributes to prostate carcinogenesis. J Exp Clin Cancer Res 40:2

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Rae JM et al (2005) GREB 1 is a critical regulator of hormone dependent breast cancer growth. Breast Cancer Res Treat 92:141–149

    Article  CAS  PubMed  Google Scholar 

  • Shioda T et al (2013) Expressomal approach for comprehensive analysis and visualization of ligand sensitivities of xenoestrogen responsive genes. Proc Natl Acad Sci USA 110:16508–16513

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Stamou M et al (2020) A balanced translocation in Kallmann syndrome implicates a long noncoding RNA, RMST, as a GnRH neuronal regulator. J Clin Endocrinol Metab 105:e23-44

    Article  Google Scholar 

  • Tan S et al (2021) Comprehensive transcriptome analysis of hypothalamus reveals genes associated with disorders of sex development in pigs. J Steroid Biochem Mol Biol 210:105875

    Article  CAS  PubMed  Google Scholar 

  • Thangavel P, Puga-Olguín A, Rodríguez-Landa JF, Zepeda RC (2019) Genistein as potential therapeutic candidate for menopausal symptoms and other related diseases. Molecules 24:3892

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Xiong J et al (2022) Genistein affects gonadotrophin-releasing hormone secretion in GT1-7 cells via modulating kisspeptin receptor and key regulators. Syst Biol Reprod Med 2022:1–13

    Google Scholar 

  • Xu Z et al (2018) miR-17-3p downregulates mitochondrial antioxidant enzymes and enhances the radiosensitivity of prostate cancer cells. Mol Ther Nucleic Acids 13:64–77

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Young MD, Wakefield MJ, Smyth GK, Oshlack A (2010) Gene ontology analysis for RNA-seq: accounting for selection bias. Genome Biol 11:R14

    Article  PubMed  PubMed Central  Google Scholar 

  • Yuen T, Ruf F, Chu T, Sealfon SC (2009) Microtranscriptome regulation by gonadotropin-releasing hormone. Mol Cell Endocrinol 302:12–17

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zhang X et al (2009) MicroRNA-17-3p is a prostate tumor suppressor in vitro and in vivo, and is decreased in high grade prostate tumors analyzed by laser capture microdissection. Clin Exp Metastasis 26:965–979

    Article  CAS  PubMed  Google Scholar 

  • Zhang Q, Bao J, Yang J (2019) Genistein-triggered anticancer activity against liver cancer cell line HepG2 involves ROS generation, mitochondrial apoptosis, G2/M cell cycle arrest and inhibition of cell migration. Arch Med Sci 15:1001–1009

    Article  CAS  PubMed  Google Scholar 

  • Zhou L et al (2010) Integrated profiling of microRNAs and mRNAs: microRNAs located on Xq27.3 associate with clear cell renal cell carcinoma. PLoS ONE 5:e15224

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zhou X et al (2016) The aberrantly expressed miR-193b-3p contributes to preeclampsia through regulating transforming growth factor-β signaling. Sci Rep 6:19910

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

This study was funded by Department of Science and Technology of Sichuan Province (Grant 2021YJ0156), the Study of Diet and Nutrition Assessment and Intervention Technology (No. 2020YFC2006300) from Active Health and Aging Technologic Solutions Major Project of National Key R&D Program, and the National Nature Science Foundation of China (Grant 82173512).

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Authors

Contributions

JX, YT and GC designed the study. JX, YT, GM and AL conducted the experiments. JX and YT analyzed and interpreted the data. JX, SS and GC provided reagents. JX and AL prepared the manuscript. JX, SS and GC reviewed and revised the manuscript. All authors have read and approved the manuscript.

Corresponding author

Correspondence to Guo Cheng.

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JX states that there is no conflict of interest to disclose. YT states that there is no conflict of interest to disclose. GM states that there is no conflict of interest to disclose. AL states that there is no conflict of interest to disclose. SS states that there is no conflict of interest to disclose. GC states that there is no conflict of interest to disclose.

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Xiong, J., Tian, Y., Ma, G. et al. Comparative RNA-seq analysis and ceRNA network of genistein-treated GT1-7 neurons. Mol. Cell. Toxicol. 19, 499–507 (2023). https://doi.org/10.1007/s13273-022-00279-1

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