沉默MAGEA6激活AMPK通路抗激素诱导成骨细胞损伤作用的机制研究

Excessive glucocorticoid (GC) usage may lead to non-traumatic femoral head osteonecrosis. Our previous studies have shown that targeted activation of AMP-activated protein kinase (AMPK) signaling could alleviate Dexamethasone (Dex)-induced osteoblast cell injuries. Our preliminary results show that melanoma antigen A6 (MAGEA6), an AMPKα1 ubiquitin ligase protein, is specifically expressed in human osteoblasts. MAGEA6 knockdown (by targeted-shRNAs) or knockout (by CRISPR-Cas9 method) induced AMPKα1 upregulation, leading to activation of AMPK and its potential downstream Nrf2, which protected osteoblasts from Dex. In addition, miR-567 is proposed to be a specific targeted MAGEA6 miRNA. miR-567 expression in human osteoblasts silenced MAGEA6 and activated AMPK. We propose that activation of AMPK by MAGEA6 silence attenuates glucocorticoid-induced osteoblast injuries. The current project will first test the expression of AMPK pathway, MAGEA6 and miR-567 in normal and GC-caused osteonecrosis femoral head tissues. Research will be performed to test how MAGEA6 silence could protect osteoblasts from Dex, and the involvement of AMPK signaling. The underlying mechanisms of AMPK-dependent Nrf2 activation by MAGEA6 silence will also be studied. The study aims to provoke AMPK activation by MAGEA6 silence in human osteoblasts, which could possibly attenuate GC-induced femoral head osteonecrosis.

地塞米松等激素可导致股骨头坏死。前期研究证实靶向激活AMPK能抑制地塞米松诱导成骨细胞损伤。预实验结果显示成骨细胞特异性表达AMPKα1泛素连接酶蛋白：黑素瘤抗原A6（MAGEA6）。而shRNA敲减或CRISPR-Cas9敲除MAGEA6则上调AMPKα1，激活AMPK及潜在下游Nrf2通路，并保护成骨细胞。此外，miR-567是MAGEA6特异性的靶miRNA。成骨细胞转染miR-567沉默MAGEA6并激活AMPK。我们假设沉默MAGEA6激活AMPK通路抗激素诱导成骨细胞损伤。本项目首先检测激素性股骨头坏死组织中AMPK通路、MAGEA6及miR-567水平；体、内外观察沉默MAGEA6或导入miR-567抗激素诱导成骨细胞损伤作用，明确激活AMPK通路在其中的作用，并解析AMPK激活Nrf2机制。旨在通过沉默MAGEA6达到激活AMPK通路并抗激素性股骨头坏死的目的。

地塞米松等激素可导致股骨头坏死。前期研究证实靶向激活AMPK能抑制地塞米松诱导成骨细胞损伤。成骨细胞特异性表达MAGEA6，导致AMPKα1泛素化降解。而通过shRNA敲减，CRISPR-Cas9 KO或miR-567靶向沉默MAGEA6，诱导成骨细胞AMPKα1表达及AMPK活化，从而抑制地塞米松诱导的成骨细胞损伤。本项目中，申请者首先检测了激素性股骨头坏死组织中AMPK信号通路蛋白（LKB1-AMPK-ACC）、MAGEA6及miR-567表达/活化水平；并运用地塞米松处理成骨细胞模型和小鼠激素性股骨头坏死模型，系统观察了MAGEA6 shRNA/KO及miR-567抗激素诱导成骨细胞损伤的作用，阐明了相关的信号转导机制，明确了激活AMPK通路在其中的作用。激素性股骨头坏死病人股骨头组织中LKB1-AMPK-ACC表达/活化水平下调，而miR-567表达水平也上调，伴随着MAGEA6上调。成骨细胞中，MAGEA6-shRNA/-KO 激活AMPK信号通路，拮抗地塞米松损伤。成骨细胞中，表达miR-567激活AMPK信号通路，拮抗地塞米松损伤。AMPK可能直接结合并磷酸化Nrf2从而激活后者，拮抗激素损伤。为激活AMPK抗激素性股骨头坏死理论提供进一步的实验证据，并为基因治疗激素性股骨头坏死提供有益探索。