Effects Of Estrogen On Osteoblast MC3T3-E1 And Its Related Mechanisms

PartⅠ:The proliferation and apoptosis of MC3T3-E1 cells were effected by estrogenObjective: In order to study the mechanism of estrogen on osteoblasts differentiation,MC3T3-E1 cells were treated with estradiol to obtain a better treatment concentration for the next step of RNA deep sequencing.Methods: We treated the mouse MC3T3-E1 cells with different concentrations of β-estradiol,then observed the morphological changes of the cells,used CCK-8 kit to detect the cell’s proliferation status and used TUNEL kit to detect apoptosis of MC3T3-E1 cells.Results: In the low-concentration treatment group,0.01 n M and 0.1 n M treated groups of the β-Estradiol treatment,we found that in the cell morphology,cell proliferation,cell apoptosis and other cell behavior compared with the DMSO control group did not produce significant difference.In the 1 n M and 10 n M,there was no significant difference in the morphology of the cells under the microscope,but there was a significant difference in cell apoptosis and proliferation.10 n M treated group appeared extremely significant differences.Conclusions: For obtaining robust results,we chose 10 n M to treat group as our deep RNA sequence experiments.PartⅡ:RNA deep sequence performed to investigate the genes and signaling pathway which induced by estrogenObjective: In order to study the mechanism of estrogen on osteoblasts,the intrinsic targeting genes and signaling pathways,we used β-estradio to treat mouse MC3T3-E1 cells and perform RNA deep sequence.Methods: We used MC3T3-E1 cells treated with estrogen.After 3 days,the cells were harvested and RNA was extracted.Two transcriptome libraries were constructed and sequenced.The results were analyzed by bioinformatics.Results: Compared with the control group,we found 460 differentially expressed genes.Among the differentially expressed genes,there were 66 up-regulated genes and 394 down-regulated genes.Using these differential expression genes,our GO classification and KEGG pathway analysis revealed that many bone metabolism related biological processes and cell signaling pathways were disordered.Conclusion: Estrogen affects cell differentiation by altering cell signaling pathways in osteoblasts.Part III Study on the molecular mechanism of estrogen on target geneObjective: The expression of Tgfbr1 and Bmpr1 a genes was significantly inhibited by estrogen in our differentially differential expression genes.Thus we study the mechanisms of estrogen inhibition of Tgfbr1 and Bmpr1 a genes.Methods: First qRT-PCR was performed to comfirmed the differentially expressed genes,and then treated the MC3T3-E1 cells with estrogen receptor α and β inhibitors,respectively,and then examined the expression of Tgfbr1 and Bmpr1 a genes.The promoter of Tgfbr1 and Bmpr1 a gene was analyzed and the estrogen receptor response elements was identified.Finally,Ch IP was used to verify the binding of estrogen receptor to Tgfbr1 and Bmpr1 a promoter.Results: The results shown that estrogen receptor beta involved in the inhibitory effect of Tgfbr1 and Bmpr1 a genes.The bioinformatics of the promoter found that there were three estrogen receptor responses elements in the promoter of Tgfbr1,and that there were two Estrogen receptor response elements in Bmpr1 a promoter regions.Ch IP experiments shown that estrogen could enhance the binding of estrogen receptors to Tgfbr1 and Bmpr1 a genes.Conclusion: Estrogen inhibits the expression of Tgfbr1 and Bmpr1 a genes through estrogen receptor β.