The Proceedings of the Eighth International Conference on Creationism (2018)

Liu, Y., and T.D. Nguyen. 2018. Integrated regulation of class II human endogenous retroviruses in a breast cancer cell line. In Proceedings of the Eighth International Conference on Creationism , ed. J.H. Whitmore, pp. 191–199. Pittsburgh, Pennsylvania: Creation Science Fellowship. INTEGRATED REGULATION OF CLASS II HUMAN ENDOGENOUS RETROVIRUSES IN A BREAST CANCER CELL LINE Yingguang Liu , Liberty University College of Osteopathic Medicine, 306 Liberty View Lane, Lynchburg, VA 24502 yliu@liberty.edu Tam D Nguyen , Liberty University College of Osteopathic Medicine, 306 Liberty View Lane, Lynchburg, VA 24502 tdnguyen5@liberty.edu ABSTRACT Endogenous retroviruses (ERVs) are still regarded as foreign invaders by most biologists. Because of structural and positional homology of ERVs in human and ape genomes, they have been considered molecular evidences of common ancestry. Using a breast cancer cell line, we analyzed the regulatory features of a group of human endogenous retroviruses (HERV-K), and found that they contain multiple sequence motifs subjecting them to regulation by sex hormones, a stem cell-specific transcription factor (OCT4), and DNA methylation. Mutation of the OCT4 motif abrogates their response to sex hormones, while methylation of a progesterone-response element enhances receptor- binding. We also found that solo LTRs of HERVK enable hormonal regulation of downstream cellular genes. The findings support the hypothesis that ERVs are integral parts of eukaryotic genomes and are designed to regulate interspersed genes, especially in reproduction and development. KEY WORDS gene regulation, cancer, progesterone, DNA methylation, long terminal repeat Copyright 2018 Creation Science Fellowship, Inc., Pittsburgh, Pennsylvania, USA www.creationicc.org 191 INTRODUCTION Creationists generally believe viruses were created for purposes beneficial to their hosts, the ecosystem, or mankind, and some became pathogenic only as a result of corruption (Bergman, 1999). Endogenous retroviruses (ERVs) are repetitive DNA elements in eukaryotic genomes that resemble DNA forms of retroviruses. Because humans and apes share similar ERVs at similar genomic locations, andERVinsertions are thought tobe largely random, ERVs have been used by evolutionists as strong evidences of common ancestry (Weiss 2006). However, during the past decade, biologists are increasingly convinced that ERVs play important functions for their host organisms, including reproduction and early embryonic development (Grow et al. 2015; Mitchell 2015). Functionality and cellular regulation of ERVs will support the creationist model that ERVs were created in the cell, and homologies between human and ape ERVs are due to functional necessity (Fabich 2015). In the Seventh ICC, we reported in vivo correlation between ERV expression and plasma levels of estradiol and progesterone in reproductive-age women (Mackey et al. 2013). Since then, we have studied the expression of various ERV elements in a human breast cancer cell line, T47D. Because T47D cells have receptors for female sex hormones, they provide an in vitro model to analyze the effects of these hormones on cellular and molecular levels. As early as in 1987, it was discovered that transcription of HERVK10 in T47D breast cancer cells is enhanced by a sequential treatment with estradiol and progesterone (Ono et al. 1987). More recent research found that female sex hormones also induce the release of retroviral particles from T47D cells (Patience et al. 1996). The goal of this project was to investigate the mechanisms of hormonal control of HERVs, which should shed light on the functional integration between the HERV elements and the rest of the genome. MATERIALS AND METHODS 1. Cell lines and culture T47D breast cancer cell line was a gift from Dr. Joseph Brewer. Cells were maintained in 10% FBS (ATLANTA Biologicals), 2 mM L-glutamine (HyClone), 1X antibiotic antimycotic solution (CORNING) containing penicillin, streptomycin, and amphotericin B, 10 mM HEPES free acid (HyClone), and basal medium RPMI 1640 (HyClone). Primary mammary epithelial cells were obtained fromATCC and were maintained in Mammary Epithelial Cell Basal Medium (ATCC) supplemented with the Mammary Epithelial Cell growth kit (ATCC) containing rH- insulin, L-glutamine, epinephrine, apo-transferrin, rH-TGFα, Extract P, and hydrocortisone hemisuccinate. 2. Extraction of RNA and reverse transcription Total cell RNAwas extracted from cultured cells using the RNeasy Plus Mini Kit (Qiagen). Complementary DNA was prepared using the Quantitect Reverse Transcription Kit (Qiagen). All RNA samples were examined for integrity using electrophoresis. Total cell RNA was incubated with the gDNA Wipeout Buffer in the reverse transcription kit for 10 minutes to eliminate residual genomic DNA. 3. Quantitative PCR The reference gene, YWHAZ, was selected using the GeNorm and NormFinder programs by comparing the stability of expression of several candidate genes in peripheral leukocytes of women during the menstrual cycle (Mackey et al. 2013). Primers for the reference gene and some HERV genes were also described in the same paper. Primers for Syncytin-2 were designed and validated by Toufaily, et al. (2015). Primers for the human pleiotrophin gene were 5’-GAGCTGAGTGCAAGCAAACC-3’ and 5’-TTCAGGGCTGTGTTCAGGTC-3’, whose product and amplification efficiency were verified by electrophoresis and quantitative PCR. Primers for SLC4A8 and IFT172 genes were designed and verified by Gogvadze et al. (2009). We used their “LTR for” as the shared forward primer, and “SLC4A8 for1” and