Bioinformatic Characteristics and Therapeutic Mechanisms of Calycosin-Anti-Bisphenol A-related Osteosarcoma

the therapeutic targets and mechanisms of calycosin-anti-BPA-related by pharmacology and docking results suggested that the biological process of the gene ontology mainly involved in ossication, positive regulation of bone resorption, positive regulation of bone remodeling, regulation of inammatory response, neuroinammatory response, positive regulation of inammatory response, interleukin-12 secretion, chronic inammatory response, positive regulation of acute inammatory response, positive regulation of interleukin-12 production, regulation of neuroinammatory response, regulation of cytokine production involved in inammatory response, cytokine production involved in inammatory response, regulation of interleukin-12 production, regulation of macrophage chemotaxis, macrophage chemotaxis, regulation of

1 Background OS, one kind of malignant bone cancers, may occur in adolescent that affect their growth and bone function [1]. Statistically, the incidence of OS is increasing yearly in China because of its huge population [2]. In medical etiology, the cause of OS may be associated with hereditary factor, food habit, and environmental exposure [3]. In recent decades, the environmental pollution is of great concerns for possible human health risk [4]. BPA, a manufactured material used widely, is a well-reported endocrine disrupting chemical that induces potential reproductive impairment, immunological and neurological dysfunctions, and tumorigenesis [5]. It is de ned by International Agency for Research on Cancer and the National Toxicology Program that BPA may be a carcinogen, such as breast cancer and prostate cancer [6]. Furthermore, BPA is found with potential risk of developing of OS in experimental studies [7][8].
However, there is no related report of agent against BPA-induced OS, especially the natural compound.
Calycosin, a naturally-occurring ingredient, is reported with functionally protecting against antioxidation, neuroprotection, anti-cancers [8]. In details, calycosin is pharmacologically found with bene cial actions against malignant cancers, such as colorectal cancer [10], hepatocellular carcinoma [11]. In our reported study shows the calycosin-anti-osteosarcoma effect in vitro, characterized with pharmacological mechanism [12]. However, the bioinformatic investigation of calycosin against BPA-induced OS remain unreported. Interestingly, a network pharmacology tool is effectively used for detection of hug biotargets and molecular mechanisms of agent to treat disease [13][14]. To attain the aim, this bioinformatic report was designed to detect and characterize network pharmacology approach to highlight all anti-BPAinduced OS targets and mechanisms of calycosin. Collectively, a ow diagram by use of network pharmacology method was planned and showed in Fig. 1 2.2 Detecting of core targets of alycosin in treatment of BPA-related OS After assays, the mapped genes of calycosin and BPA-related OS were used to generate a protein-protein interaction (PPI) network of calycosin to treat BPA-related OS using STRING tool. And the tsv. data were determined via Cytoscape tool to collect the core targets. In details, the core targets were screened according to the Degree value using NetworkAnalyzer for the topological data [17][18]

Construction of network visualization
Applying with Cytoscape software to assay the core targets, the data of gene ontology of biological process and pathway enrichment for calycosin to treat BPA-related OS. Further, the visualization graph of drug-target-gene ontology-biological process-pathway-disease was constructed [21][22].

Molecular docking veri cation
As reported previously [23], all core targets were veri ed by molecular docking, and the structure of calycosin compound was obtained from PubChem database. The related protein structure was collected from the PDB database. Applying with Chem Bio O ce2010 software, the docked ligand molecule and the original ligand molecule, the rationality of the docking parameter setting can be determined and identi ed according to the root mean square deviation (RMSD). It was generally referenced that RMSD ≤ 4 Å was the threshold for the conformation of the ligand to match the conformation of the original ligand after molecular docking.

Preliminary bioinformatics data of targets
After tests by online tools, the number of 245 BPA-related OS disease-genes were detected, and analytical 138 anti-disease genes of calycosin were determined. As shown in Venn graph, a total of 20 mutual genes of calycosin and BPA-related OS were identi ed before being highlighted in a interaction network of calycosin to treat BPA-related OS (Fig. 1A).

Findings of all core targets
The mutual genes were further assayed by Cytoscape software, and then the topological data showed that the median degree of freedom of the target was 3.667, and the maximum degree of freedom was 10.
Therefore, the core target screening criteria range was set to 4-10. As a result, we identi ed total 9 core targets of calycosin to treat BPA-related OS, having EGFR, ESR1, HSP90AA1, MAPK14, ESR2, AR, BRCA1, PTGS2, CYP19A1. The ndings illustrated in an interaction network (Fig. 1B) and Supplemental Table 1. 3.3 Enrichment analysis ndings of core targets All 9 core targets were used to determine the gene ontology enrichment and KEGG pathway enrichment analyses through R language-related packages. As results, the histogram and Circos circle chart of the biological process of the gene ontology were presented in Figure. 3A-B; and then the histogram and Circos circle chart of KEGG pathways were showed in Figure. 3C-D. The results suggested that the biological process of the gene ontology mainly involved in ossi cation, positive regulation of bone resorption, positive regulation of bone remodeling, regulation of in ammatory response, neuroin ammatory response, positive regulation of in ammatory response, interleukin-12 secretion, chronic in ammatory response, positive regulation of acute in ammatory response, positive regulation of interleukin-12 production, regulation of neuroin ammatory response, regulation of cytokine production involved in in ammatory response, cytokine production involved in in ammatory response, regulation of interleukin-12 production, regulation of macrophage chemotaxis, macrophage chemotaxis, regulation of macrophage migration, macrophage migration, negative regulation of macrophage migration, positive regulation of macrophage chemotaxis, regulation of cytokine secretion involved in immune response, cytokine secretion involved in immune response, positive regulation of macrophage migration, neutrophil activation involved in immune response, neutrophil mediated immunity, negative regulation of leukocyte migration, negative regulation of I-kappaB kinase/NF-kappaB signaling, positive regulation of cytokine production involved in immune response, response to tumor necrosis factor, response to antineoplastic agent, regulation of signal transduction by p53 class mediator, signal transduction by p53 class mediator, cellular response to tumor necrosis factor, response to steroid hormone, intracellular estrogen receptor signaling pathway, cellular response to steroid hormone stimulus, intracellular steroid hormone receptor signaling pathway, regulation of intracellular estrogen receptor signaling pathway, steroid hormone mediated signaling pathway, hormone-mediated signaling pathway (Supplemental Table 2). The 26 KEGG pathways of the core targets (P < 0.05) were identi ed with involvement of IL-17 signaling pathway, Th17 cell differentiation, Endocrine resistance, Estrogen signaling pathway, Prolactin signaling pathway, Ovarian steroidogenesis, Progesterone-mediated oocyte maturation, Breast cancer, Prostate cancer, Proteoglycans in cancer, MicroRNAs in cancer, PD-L1 expression and PD-1 checkpoint pathway in cancer, VEGF signaling pathway, GnRH signaling pathway, C-type lectin receptor signaling pathway, PI3K-Akt signaling pathway, TNF signaling pathway, Relaxin signaling pathway, FoxO signaling pathway, Oxytocin signaling pathway (Supplemental Table 3).

Interaction network ndings
The network visualization of calycosin-target-BP-KEGG-BPA/OS was determined and highlighted through Cytoscape software, as revealed in Figure. 4. The core targets enriched in the KEGG pathway were identi ed in red by R-language software, as detailed in Fig. 5.

Discussion
In this bioinformatics study, we identi ed and elucidated the pharmacological activity and mechanism of calycosin to treat BPA-related OS. More interestingly, all core targets of calycosin to treat BPA-related OS were revealed, comprising EGFR, ESR1, HSP90AA1, MAPK14, ESR2, AR, BRCA1, PTGS2, CYP19A1. EGFR, if mutated, is reportedly found with the development of tumorigenesis [24]. Another evidence shows that BPA induces cancer cell proliferation via activation of EGFR activity [25]. ESR1, also known as estrogen receptor 1, is commonly found with mutation in many human cancer samples, such as breast cancer [26]. It is reported that high BPA exposure may be related to non-small cell lung cancer via changing ESR1 genetic polymorphism [27]. It is also reported in the literatures that in addition to affecting tumor cell survival, HSP90AA1 can act on the invasion and migration of cancer cells, and it is closely related to the poor prognosis of tumors [28]. HSP90AA1, functioning as a key effector of autophagy, is positively associated with the development of osteosarcoma chemoresistance [29]. MAPK14 acts as an integration point for multiple biochemical events, and it is involved in a group of cellular processes including cell differentiation and proliferation, gene transcription regulation [30]. Some of evidences suggest that MAPK14 activity activates the cancer cell migration, invasiveness and angiogenesis [31]. AR, an androgen receptor, is a drug target in cancers, such as prostate tumor, and the anti-cancer action is achived through regulating androgen receptor signaling [32]. It is reported that BPA regulates speci c gene expression in human prostate cancer cells for androgen-dependent proliferation [33]. BRCA1, a nuclear phosphoprotein, exerts an action in maintaining genomic stability, and it also functions as a cancer suppressor [34]. It is found that BRCA1 may suppress the BPA-induced cell proliferation in vitro and in vivo [35]. The transcriptional pro ling of PTGS signaling characterizes the key clue on the biological event of the tumor-related microenvironment, such as in ammatory in ltration [36]. However, there is no report regarding the association between PTGS and osteosarcoma, BPA. The clinical analysis suggests the potential impact of CYP19A1 in postmenopausal endocrine responsive breast cancer [37]. The study in vitro indicates that BPA induces cell proliferation and growth in human choriocarcinoma cell line [38]. As revead in computational assaus, the pharmacological functions of to treat BPA-related OS were identi ed accordingly, such as ossi cation, positive regulation of bone resorption, positive regulation of bone remodeling, regulation of in ammatory response, neuroin ammatory response, positive regulation of in ammatory response, interleukin-12 secretion, positive regulation of interleukin-12 production. These bioinformatic ndings indicate that calycosin may play the anti-BPA-related OS action by modulating current molecular processes. In further investigation, the other KEGG enrichment analysisbased ndings revealed all calycosin-anti-BPA-related OS, including IL-17 signaling pathway, Th17 cell differentiation, Endocrine resistance, Estrogen signaling pathway, Proteoglycans in cancer, MicroRNAs in cancer, PD-L1 expression and PD-1 checkpoint pathway in cancer, VEGF signaling pathway, GnRH signaling pathway PI3K-Akt signaling pathway, TNF signaling pathway, FoxO signaling pathway. More markedly, our current computational evidences of GO-based molecular processes were consistent with the ndings of KEGG signaling pathways of calycosin against BPA-related OS following bioinformatic analyses, contributing to the future clinical application of calycosin against BPA-related OS.

Conclusion
In conclusion, our current computational report highlights the pharmacological biotargets, biological processes, molecular mechanisms of calycosin to treat BPA-related OS. In future medical signi cance, current bioinformatic ndings will be applied in clinical practice against osteosarcoma, including BPArelated OS.