ESR1, also known as ER-alpha or ERα, is an important estrogen receptor (ER) and involves in the gene regulation in various diseases and biological processes, including breast cancer (1,2), osteosarcoma (3) and cell growth (4). 17β-estradiol (E2) is one of the most representative estrogens responsible for the development and reproductive capability of female characteristics (5). Besides, it participates in the progression of many diseases, for example, E2 could regulate the proliferation of breast cancer cells through focal adhesion and chemokine signaling pathways (6); through autophagy and apoptosis pathways, E2 plays important roles in the regulation of renal cell carcinoma growth (7). Furthermore, ER, including ERα and ERβ, are important media through which E2 involves the regulation of many biological processes. So, exploration of interactions between ER and E2 would promote our understanding of many types of complex diseases and the identification of their potential therapeutic targets.

Osteosarcoma is one of the most common malignant neoplasms in children and adolescents with ~20% 5-year overall survival rate after chemotherapy (8–10). In primary osteosarcoma, adequate surgical margins could significantly improve the prognosis, whereas, it is still a challenging surgical technique (11). Moreover, metastasis greatly affects prognosis for osteosarcoma due to the propensity of early metastatic trend and ~80% of osteosarcoma patients have metastasis at diagnosis (12,13). Understanding its mechanisms and screening of potential biomarkers would be helpful for its early diagnosis and improvement of prognosis. The development of biotechnologies have promoted the identification of diagnosis and therapeutic targets for osteosarcoma. For example, microRNA-491-5p was considered to suppress cell proliferation and promote apoptosis via FOXP4 in the report of Yin et al (14) through the quantification of miR-491-5p expression and cell apoptosis; through immunohistochemistry and statistic analysis, Zhao et al (15) identified HIF1 as an independent prognostic biomarker in osteosarcoma; furthermore, Zhou et al (16) even indicated that the level of miR-199a-5p in serum/plasma might serve as biomarker for osteosarcoma.

Estrone and estriol, also known as E1 and E3, are another type of estrogens which have been proved to be involved in the progression of several diseases, including osteosarcoma. For example, Mochizuki et al proved that E3 is compatible with E2 to induce the expression of IGF-1 mRNA and then affect the proliferative potential of osteosarcoma cells (17). Besides, E1 sulfate represents the most abundant estrogen in circulation of adults and plays a role in human bone maturation and homeostasis (18), and it has been shown to influence the progression of many types of cancers, such as colorectal cancer (19) and breast cancer (20), so it might be also associated with the development of osteosarcoma.

There are also several studies on E2 and ESR1 in osteosarcoma (3,21,22). While, no study has focused on the genome binding profiles of ESR1 with the presence of E2 and the E2-regulated genes through ESR1 simultaneously. In this study, a combined analysis of ESR1 chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) with the presence of E2 and gene expression profiles of E2-regulated through ESR1 was conducted. This would induce several potential target genes of ESR1 in osteosarcoma, and here, the E2-regulated genes in balance. This would be helpful for the identification of E2 targeted molecules in osteosarcoma and the development of corresponding drugs.

Osteosarcoma is the most common bone tumor with high morbidity and mortality. E2 was found to play important roles in osteosarcoma through regulating physiological processes and gene expression via ESR1 by many studies (3,32). While, the combined analysis of E2-mediated gene expression and regulation is still limited. In this study, we conducted gene microarray and ChIP-seq analysis in E2 treated U2OS-ERα cell lines, which would be useful to promote our understanding of the E2-mediated gene regulation in osteosarcoma.

E2-activated ESR1 could directly or indirectly bind to its target genes and then regulate the particular biological processes or gene expression. In osteosarcoma, E2 is necessary for the translocation of ESR1 to the nuclei. For example, Hu et al showed that ESR1 could promote the binding of Sp1 to the promoter of MALAT1, a long non-coding RNA, in U2OS cells to affect physiological processes only with E2 stimulation (32); Tübel et al even proved that the methylation status of ESR1 was affected by E2 (3). In this study, the ChIP-seq analysis of E2 stimulated U2OS-ERα cell identified the potential target genes of E2 mediated through ESR1. Besides, cross analysis with gene microarray could further screen out more reliable target genes for osteosarcoma.

Functional enrichment analysis resulted in the identification of cancer, cell activity-related KEGG pathways, which further confirmed our results. Estrogen signaling pathway was found to be significantly enriched in DEG4 and DEG48, which indicated roles of E2 in the activation of ESR1.

E2 could also regulate the expression level of microRNA (miRNA) and affect cell proliferation, migration and invasion in osteosarcoma. In turn, E2-mediated genes might also be regulated by miRNA. In this study, we performed miRNA-Gene regulation analysis for overlaps between PGenes and overlapped genes among the four lists of DEGs. Finally, 12 out of the 15 overlaps were found to be regulated by miRNA and ACC1 with the highest degree. ACC1 could act as anti- and pro-apoptotic regulators which involves in several cellular activities, including embryonic development, homeostasis, and tumorigenesis. Besides, it has been proved as potential biomarker in many types of cancers, such as gliomas, and melanoma (33,34). Significant enrichment of ESR1-specific binding peaks were identified in the promoter of WNK1 (Fig. 4, bottom), which was regulated by 31 miRNAs in this study. WNK1 and ESR1 were found to have differential expression in osteoporosis simultaneously (35). GREB1 was found with differential expression in U2OS-ERα cells at 4, 8, 24 and 48 h after E2 treated and regulated by E2 through ESR1. Consistent with our study, Lin and Lei reported that GREB1 was a estrogen-responsive gene and with two canonical estrogen response elements (36).

In conclusion, this study identified the potential E2-mediated genes through ESR1 in osteosarcoma via the combined analysis of ChIP-seq and gene microarray analysis. It could be helpful for the identification of therapeutic targets for osteosarcoma.