Abstract
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 effect of genistein on bone differentiation, RNA sequencing (RNA-seq) analysis was used to detect differentially expressed genes (DEGs) in genistein-treated vs. untreated MC3T3-E1 mouse osteoblastic cells. Osteoblastic cell differentiation was monitored by measuring osteoblast differentiation factors (ALP production, bone mineralization, and expression of osteoblast differentiation markers). From RNA-seq analysis, a total of 132 DEGs (including 52 up-regulated and 80 down-regulated genes) were identified 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 differentiation 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 differentiation, while three down-regulated genes (Hrc, Gli, and Ifitm5) suppress the differentiation. These results imply their major functional roles in bone differentiation regulated by genistein.
Highlights
Genistein, a phyto-estrogen, can potentially replace endogenous estrogens in postmenopausal women, but the underlying molecular mechanisms remain incompletely understood
MTT, Alkaline phosphatase (ALP), and Alizarin red S staining assays detected the maximum expression of markers of osteoblastic differentiation, without severe cell death, at a genistein concentration of 10 μM (Supplementary Figs 1–3)
Because the MC3T3-E1 cell line expresses estrogen receptors and can differentiate into osteoblasts, it has been a useful model for studying the molecular mechanism of osteogenesis[26]
Summary
A phyto-estrogen, can potentially replace endogenous estrogens in postmenopausal women, but the underlying molecular mechanisms remain incompletely understood. These siRNA knockdown experiments (of the DEGs validated by real-time qPCR) demonstrated that two up-regulated genes (Ereg and Efcab2) enhance osteoblastic cell differentiation, while three down-regulated genes (Hrc, Gli, and Ifitm5) suppress the differentiation These results imply their major functional roles in bone differentiation regulated by genistein. The discovery of alternative agents that could replace estrogen would be highly beneficial for skeletal and overall health Candidates for this purpose include phytoestrogens, plant-derived compounds with structures and functions similar to those of mammalian estrogens. Their use as alternative treatments for osteoporosis, potentially lacking some or all of the side effects of estrogen, has been extensively studied[14]. Genistein likely affects 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]
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