Estrogen receptor β induces autophagy of osteosarcoma through the mTOR signaling pathway

Zhengming Yang1* , Wei Yu1 , Bing Liu1 , Minfei Yang2 and Huimin Tao1

Abstract Background: Estrogen receptor beta (ERβ) was considered as a tumor-inhibiting factor in estrogen-sensitive malignant tumors. In this study, we intended to investigate whether ERβ was involved in inducing autophagy in osteosarcoma. Methods: This is an experimental study. The associations between ERβ and autophagy were detected in osteosarcoma U2-OS cells which were treated with E2, E2 + 2,3-Bis (4-hydroxyphenyl) propionitrile (DPN, ERβ agonists), E2 + DPN + water, E2 + DPN + 3-Methyladenine (3-MA, autophagy inhibitor), respectively. Cell viability and death were detected using cell counting kit 8 assay and flow cytometry, respectively. In addition, the expression of autophagy marker LC3II/I, sequestosome 1 (P62), mammalian target of rapamycin (mTOR), and phosphorylated-mTOR (p-mTOR) was determined by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blotting. Results: Cell viability was significantly decreased with DPN treatment, while was reversed with 3-MA treatment. DPN treatment decreased living cells proportion and increased cell apoptosis proportion, while 3-MA treatment reversed those changes. However, there were significant differences between the E2 group and the E2 + DPN + 3- MA group for the living cell proportion and cell apoptosis proportion, suggesting apoptosis and autophagy all were induced. In addition, DPN treatment upregulated the LC3II/I expression level and downregulated P62 and mTOR (mRNA level) and p-mTOR (protein level) expression levels. Conclusion: ERβ inhibited the cell viability and mediated cell death by inducing apoptosis and autophagy in osteosarcoma. ERβ-induced autophagy in osteosarcoma was associated with downregulating the P62 expression level and inhibiting mTOR activation. Keywords: Osteosarcoma, Estrogen receptor beta, Autophagy, mTOR.

详情请见：https://doi.org/10.1186/s13018-020-1575-1

在该研究中圣尔生物通用型ECL发光液用于Western blotting实验

Western blotting

After washing with PBST buffer six times, the membrane was exposed with enhanced chemiluminescence (ECL) reagents (catalog no. SB-WB012, Share-bio) and the Millipore ECL system, and then gray scanning was performed using TanonImage.