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Understanding the Functional Role of Genistein in the Bone Differentiation

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Understanding the Functional Role of Genistein in the Bone Differentiation www.nature.com/scientificreports Correction: Author Correction OPEN Understanding the functional role of genistein in the bone diferentiation in mouse Received: 16 June 2017 Accepted: 7 February 2018 osteoblastic cell line MC3T3-E1 by Published online: 19 February 2018 RNA-seq analysis Myungsuk Kim1,2, Jisun Lim3, Jung-Hee Lee3, Kyung-Mi Lee1,2, Suji Kim4, Kye Won Park4, Chu Won Nho1,2 & Yoon Shin Cho3 Genistein, a phyto-estrogen, can potentially replace endogenous estrogens in postmenopausal women, but the underlying molecular mechanisms remain incompletely understood. To obtain insight into the efect of genistein on bone diferentiation, RNA sequencing (RNA-seq) analysis was used to detect diferentially expressed genes (DEGs) in genistein-treated vs. untreated MC3T3-E1 mouse osteoblastic cells. Osteoblastic cell diferentiation was monitored by measuring osteoblast diferentiation factors (ALP production, bone mineralization, and expression of osteoblast diferentiation markers). From RNA-seq analysis, a total of 132 DEGs (including 52 up-regulated and 80 down-regulated genes) were identifed in genistein-treated cells (FDR q-value < 0.05 and fold change > 1.5). KEGG pathway and Gene Ontology (GO) enrichment analyses were performed to estimate the biological functions of DEGs and demonstrated that these DEGs were highly enriched in functions related to chemotactic cytokines. The functional relevance of DEGs to genistein-induced osteoblastic cell diferentiation was further evaluated by siRNA-mediated knockdown in MC3T3-E1 cells. These siRNA knockdown experiments (of the DEGs validated by real-time qPCR) demonstrated that two up-regulated genes (Ereg and Efcab2) enhance osteoblastic cell diferentiation, while three down-regulated genes (Hrc, Gli, and Iftm5) suppress the diferentiation. These results imply their major functional roles in bone diferentiation regulated by genistein. Bone structure is regulated by a dynamic balance between bone formation mediated by osteoblasts and bone resorption mediated by osteoclasts1,2. Osteoblasts, which arise by diferentiation from mesenchymal stem cells (MSC), play important roles in osteogenesis3, and the continuous diferentiation of MSCs causes osteoblasts to undergo maturation and mineralization3,4. Bone formation process proceeds through interactions among various proteins5,6. For example, osteoblastogenesis is regulated through the activation of crucial transcription factors such as Runt-related transcription factor 2 (RUNX2), along with upregulation of osteogenic proteins including alkaline phosphatase (ALP, an early stage osteoblast diferentiation marker), osteopontin (OPN, a mineralization inhibitor), and osteoprotegerin (OPG, an inhibitor of osteoclast diferentiation)7,8. RUNX2 is itself activated by the BMP signaling pathway, and thus bone morphogenetic protein 2 (BMP2) is a master regulator of osteoblast diferentiation9. Te expression of proteins involved in the regulation of osteoblast diferentiation can be infu- enced by various factors, including growth factors, estrogen, and cytokines6,10,11. Estrogen, one of the sex steroid hormones, is frequently used in hormone therapy because of its importance in maintaining bone mass12. Despite its extensive use in treating osteoporosis, estrogen has various side efects, 1Convergence Research Center for Smart Farm Solution, Korea Institute of Science and Technology, Gangneung, 25451, Republic of Korea. 2Natural Products Research Center, Korea Institute of Science and Technology, Gangneung, 25451, Republic of Korea. 3Department of Biomedical Science, Hallym University, Chuncheon, 24252, Republic of Korea. 4Department of Food Science and Biotechnology, Sungkyunkwan University, Suwon, 16419, Republic of Korea. Myungsuk Kim and Jisun Lim contributed equally to this work. Correspondence and requests for materials should be addressed to C.W.N. (email: [email protected]) or Y.S.C. (email: [email protected]) SCIENTIFIC REPORTS | (2018) 8:3257 | DOI:10.1038/s41598-018-21601-9 1 www.nature.com/scientificreports/ Figure 1. Heatmap of top 15 up- and 15 down-regulated genes in genistein treated mouse osteoblastic cell line MC3T3-E1. including elevated risks of certain cancers13. Tus, the discovery of alternative agents that could replace estrogen would be highly benefcial for skeletal and overall health. Candidates for this purpose include phytoestrogens, plant-derived compounds with structures and functions similar to those of mammalian estrogens. Teir use as alternative treatments for osteoporosis, potentially lacking some or all of the side efects of estrogen, has been extensively studied14. Genistein is a non-steroidal phytoestrogen found in various plants, including soy, fava, and lupin15. Because it is structurally similar to 17β-estradiol, genistein is considered as a potential therapeutic agent for preventing postmenopausal osteoporosis16. Genistein inhibits bone loss in part by promoting bone formation17, i.e., it contributes to maintenance of bone mass not only by decreasing osteoclastogenesis but also by increasing osteoblastogenesis18–20. A previous clinical study reported that intake of soy isofavone extracts, including genistein, induces stimulation of osteoblast function in postmenopausal women21. Genistein afects several molecular pathways involved in stimulating osteogenic bone formation and inhib- iting osteoclastic bone resorption. Genistein induces the expression of ALP, bone sialoprotein (BSP), OCN, and osteopontin (OPN) by activating the p38MAPK–Runx2 pathway, which promotes osteogenesis22. Genistein also stimulates proliferation and osteoblastic diferentiation through the nitric oxide/cGMP pathway22,23. To inhibit osteoclastic bone resorption, genistein acts as an inhibitor of tyrosine kinases22. Genistein likely afects the NF-ĸB pathway, which promotes osteoclast formation, via the activities of RANKL (receptor activator of NF-ĸB ligand), RAN (receptor activator of NF-ĸB), and OPG (osteoprotegerin)22,24. Although several molecular mechanisms of genistein have been explored in bone cells, its effect on genome-wide gene regulation involved in bone formation and resorption remains largely unknown. To under- stand the global molecular mechanisms of genistein in bone diferentiation, we conducted RNA sequencing (RNA-seq) analyses in genistein-treated and untreated MC3T3-E1 mouse osteoblastic cells. We also sought to identify novel genes that are associated with the osteogenic responses of MC3T3-E1 cells to genistein treatment. Results Identifcation of DEGs in genistein-treated cells by RNA-seq. With the goal of understanding the efect of genistein on bone diferentiation, we performed polyadenylated paired-end RNA-seq analysis to identify DEGs in genistein-treated vs. untreated MC3T3-E1 cells. Prior to this experiment, we optimized the dose of genistein for induction of bone diferentiation. MTT, ALP, and Alizarin red S staining assays detected the maxi- mum expression of markers of osteoblastic diferentiation, without severe cell death, at a genistein concentration of 10 µM (Supplementary Figs 1–3). Based on these fndings, 10 µM genistein was administered to MC3T3-E1 cells in both the RNA-seq experiments and subsequent functional studies, described below. Approximately 311 million paired-end sequencing reads were obtained from six samples (three genistein-treated and three untreated). About 85% of sequencing reads could be mapped to the reference mouse genome using the TopHat and Bowtie sofware (Supplementary Table 4). Using Cufinks, mapped reads were assembled to the genome, and genome-wide gene expression levels were determined on the basis of the FPKM normalization. Cuffdiff analysis revealed a total of 132 DEGs (52 up-regulated and 80 down-regulated) in genistein-treated cells that fulflled our DEG selection criteria, adjusted p-value (reported as q-value in Cufdif) <0.05 and fold change >1.5 (Supplementary Table 5 and Supplementary Fig. 4). Of 132 DEGs, the 15 up- and 15 down-regulated genes with the largest fold changes in MC3T3-E1 cells (Fig. 1) were selected for the validation by qRT-PCR assays. Of these 30 selected DEGs, we measured the mRNA levels of 20 (11 up-regulated and 9 down-regulated, excluding ten functionally uncharacterized genes) in the presence and SCIENTIFIC REPORTS | (2018) 8:3257 | DOI:10.1038/s41598-018-21601-9 2 www.nature.com/scientificreports/ Figure 2. Efect of genistein on mRNA expression of selected diferentially expressed genes in MC3T3-E1 cells. (A) Analysis of mRNA on up-regulated selected genes (Ccl7, Lif, Mmp-13, Wisp2, Ereg, Il6, Pde4b, Katnal2, Efcab2, Gdnf) in RNA sequencing. (B) Analysis of mRNA on down-regulated selected genes (Camk2b, Cxcl9, Gli1, Hrc, Iftm5, Klhl30, Cngb1, Rhbdl2, Susd4) in RNA sequencing. Specifc mRNA expression values were normalized to the expression of β-actin. Results are expressed as mean ± S.D of three independent experiments. (*P < 0.05, **P < 0.01 compared with control group). absence of genistein. About 70% of qRT-PCR tested genes exhibited expression patterns similar to those observed in RNA-seq experiments (Fig. 2A–B and Table 1). Genistein-regulated expression of these genes was further con- frmed in mouse bone marrow-derived osteoblasts (Supplementary Fig. 5). Determination of biological roles of DEGs by in silico methods. To obtain insight into the overall biological efect of genistein on mouse osteoblastic cells, we performed KEGG pathway analysis of the DEGs detected in this study. Of 132 DEGs, 127 were strongly enriched (Benjamini p-value < 0.05)
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