# Transcriptome Analysis of Egg Yolk Sialoglycoprotein on Osteogenic Activity in MC3T3-E1 Cells
## 4 Discussion  
Recently, several studies used transcriptional profiling to address osteoblast differentiation. Some focus on gene expression profile of osteoporotic phenotype mice [41], and others used transcriptional analysis to investigate gene expression differences in osteoblast differentiation of MC3T3-E1 cells [42; 43]. In this study, after we identified several common pathways and connected DEGs with potential biological pathways by transcriptome analysis, we found that EYG had an effect on several bone formation related pathways, such as TNF signaling pathway, complement and coagulation cascades, protein digestion and absorption, ECM-receptor interaction and focal adhesion. In our study, we noticed the TNF signal pathway and complement and coagulation cascades were downregulated with the EYG treatment. It has been proved that complement and coagulation cascades are activated after bone fracture, triggering a local or systemic inflammatory response [44]. Besides, persistent inflammation can control bone mass by affecting bone formation balance, and TNF-\(\alpha\) as one of the most potential pro-inflammatory cytokines can inhibit osteoblast differentiation and bone regeneration under the inflammatory conditions [45; 46]. As for the inflammatory factors in the pathway, BCL3 and NOD2 can activate the downstream NF-\(\kappa\)B signaling to promote RANKL-induced osteoclastogenesis [47; 48]. Modulating imbalance and dysregulation of proinflammatory mediators CCL5 can improve bone formation [49] and high expression of CCL2 is often associated with macrophage aggregation and inflammatory response [50]. On this basis, EYG may  
Figure 7: Gene relative expressions of COL-I (**A**), BMP2 (**B**), Runx2 (**C**) and \(\beta\)-catenin (**D**) by qRT-PCR analysis. MC3T3-E1 cells were treated with various concentrations of EYG. EYG significantly increased the above key factors of MC3T3-E1 osteoblast. Data were expressed as mean \(\pm\) SEM and the number of replicates was 3 (\(n=3\)). Multiple comparisons were done using one way ANOVA analysis. \({}^{*}\)\(p<0.05\), \({}^{**}\)\(p<0.01\) versus control. inhibit the gene expression of osteoclasts and promote the formation of osteoblasts by downregulating inflammatory factors. In the current study, ECM-receptor interaction, focal adhesion and protein digestion and absorption were the most significant pathways for upregulated genes. ECM, as an important component of cell microenvironment, influences cell behavior and tissue development by regulating cell adhesion, migration, apoptosis, proliferation and differentiation [51,52]. Tissue-specific and stage-specific ECM plays an important role in regulating differentiation of mesenchymal stem cells into specific cell types [53]. Focal adhesion is also closely related to osteoblastic differentiation. Laminin-5 can make focal adhesion kinase (FAK) activation regulate osteogenic differentiation of hMSCs without any soluble osteogenic supplements [54]. Moreover, transmembrane integrin heterodimers are key factors in focal adhesion pathways, connecting extracellular domains to ECM molecules such as collagen, thus playing a role in osteoblast differentiation [55]. ECM proteins include collagen and non-collagenous glycoproteins [56,57]. In addition to COL-I, there are many collagen proteins that are closely related to calcification deposition in osteoblastic differentiation. COL2A1 served as markers of the differentiation of proliferative chondrocytes [58]. COL4 is one of the main components of cellular basement membranes and COL4A1 and COL4A2 encode the \(\alpha\)1, \(\alpha\)2 chain of COL4. Mutations in COL4A1 and COL4A2 may lead to maladjustment of multiple systems, resulting in a variety of multifactorial diseases [59]. Microarray analysis proved that COL4A1 mRNA has more expression in osteoporoptic human bone than in normal human bone [60].