Background: Estrogen, as an important hormone in human physiological process, is closely related to bone metabolism. The aim of this study was to investigate the mechanism of estrogen on osteoblasts metabolism in MC3T3-E1 cells.
Methods: We treated the MC3T3-E1 cells with different concentrations of β-estradiol (0.01, 0.1, 1, and 10 nmol/L), observed the morphological changes of the cells, and detected the cell's proliferation and apoptosis of MC3T3-E1 cells. Two transcriptome libraries were constructed and sequenced. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to confirm the differentially expressed genes (DEGs), and then treated the MC3T3-E1 cells with estrogen receptor (ER) inhibitors α and β, respectively, and then examined the expression of Tgfbr1 and Bmpr1a genes. The promoter of Tgfbr1 and Bmpr1a gene was analyzed, and the ER response elements were identified. Finally, ChIP was used to verify the binding of ER to Tgfbr1 and Bmpr1a promoter.
Results: In the high-concentration β-estradiol treatment group (1 nmol/L and 10 nmol/L), there was no significant difference in the morphology of the cells under the microscope, 1 nmol/L and 10 nmol/L treated group appeared statistically significant difference in cell apoptosis and proliferation (P < 0.05 and P < 0.01, respectively). We found 460 DEGs compared with the control group. Among the DEGs, there were 66 upregulated genes and 394 downregulated genes. Gene ontology classification and Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed that many bone metabolism-related biological processes and cell signaling pathways were disordered. The qRT-PCR verification showed that the expressions of Tgfbr1- and Bmpr1a-related genes in bone metabolism pathway in the 10 nmol/L treatment group were significantly decreased (P < 0.05). ER β was involved in the inhibitory effect of Tgfbr1 and Bmpr1a genes. The bioinformatics of the promoter found that there were three ER response elements in the promoter of Tgfbr1, and there were two ER response elements in Bmpr1a promoter regions. ChIP experiments showed that estrogen could enhance the binding of ERs to Tgfbr1 and Bmpr1a genes.
Conclusions: Estrogen can promote the apoptosis and proliferation of osteoblasts simultaneously, and the mechanism may be the joint action of transforming growth factor-beta, Wnt, mitogen-activated protein kinase, and nuclear factor-kappaB bone metabolism-related signaling pathway. Estrogen inhibits the expression of Tgfbr1 and Bmpr1a genes through ER β and affects the metabolism of MC3T3-E1 osteoblasts.
雌激素通过雌激素受体β抑制MC3T3-E1细胞Tgfbr1和Bmpr1a基因的表达 摘要 背景：雌激素作为人体生理过程中重要的激素，与骨代谢密切相关。本研究利用MC3T3-E1作为细胞模型，进一步研究雌激素对骨代谢的影响。 方法：分别用0.01、0.1、1和10 nmol/L浓度的β-雌二醇处理成骨细胞MC3T3-E1，并设对照组；然后利用CCK-8试剂盒、TUNEL试剂盒检测不同浓度下MC3T3-E1细胞的增值和凋亡状况；选用10 nmol/L β-雌二醇的处理组，与对照组构建两个转录组库，进行RNA深度测序分析；利用qRT-PCR验证差异表达的基因，使用雌激素受体抑制剂分别处理MC3T3-E1细胞，检测Tgfbr1和Bmpr1a两个基因的表达。启动子分析软件分析Tgfbr1和Bmpr1a基因，ChIP实验验证雌激素受体β对Tgfbr1和Bmpr1a启动子的绑定作用。 结果：在低浓度（0.01 nmol/L和0.1 nmol/L）的处理组中，在细胞的形态、细胞的增殖和细胞的凋亡等细胞行为方面相对于DMSO对照组都没有产生明显的差异（P>0.05）；在1 nmol/L和10 nmol/L高浓度β-雌二醇处理组中，细胞的凋亡和细胞的增殖活性增强，1 nmol/L处理组有明显差异（P<0.05），并且在10 nmol/L处理组出现了极其显著的差异（P<0.01）。深度测序发现10 nmol/L处理组中有460个差异表达基因，在这些差异表达基因中，上调的基因有66个，下调的基因有394个。利用这些差异表达基因，本研究进行GO分类和pathway分析，发现许多骨代谢相关的生物过程和细胞信号通路。在这些被富集的骨代谢相关的信号通路中，包括经典的TGF-β信号通路，Wnt信号通路，MAPK信号通路。差异表达基因qRT-PCR验证显示，10 nmol/L 处理组中骨代谢通路相关基因Tgfbr1和Bmpr1a表达明显下降（P<0.05）。雌激素受体β参与了雌激素对Tgfbr1和Bmpr1a基因的抑制性作用，生物信息学分析发现，在Tgfbr1的启动子区域存在3个雌激素受体应答原件，在Bmpr1a的启动子区域有2个雌激素受体应答原件。ChIP实验证明， 雌激素能够增强雌激素受体β对Tgfbr1和Bmpr1a基因的绑定性作用。 结论：雌激素能同时促进成骨细胞凋亡、增殖， 其机制可能是TGF-β、Wnt、MAPK及NF-kappaB骨代谢相关信号通路共同作用结果；雌激素通过雌激素受体β抑制Tgfbr1和Bmpr1a基因的表达， 影响MC3T3-E1成骨细胞代谢。.
Keywords: Bmpr1a; Estrogen; Estrogen Receptor Beta; MC3T3-E1 Cells; Tgfbr1.