Ye Xueting, Xie Jing, Huang Hang, Deng Zhexian
Department of Urology, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China.
Department of Stomatology, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China.
Cell Physiol Biochem. 2018;45(3):1205-1218. doi: 10.1159/000487452. Epub 2018 Feb 9.
BACKGROUND/AIMS: Melanoma antigen A6 (MAGEA6) is a cancer-specific ubiquitin ligase of AMP-activated protein kinase (AMPK). The current study tested MAGEA6 expression and potential function in renal cell carcinoma (RCC).
MAGEA6 and AMPK expression in human RCC tissues and RCC cells were tested by Western blotting assay and qRT-PCR assay. shRNA method was applied to knockdown MAGEA6 in human RCC cells. Cell survival and proliferation were tested by MTT assay and BrdU ELISA assay, respectively. Cell apoptosis was tested by the TUNEL assay and single strand DNA ELISA assay. The 786-O xenograft in nude mouse model was established to test RCC cell growth in vivo.
MAGEA6 is specifically expressed in RCC tissues as well as in the established (786-O and A498) and primary human RCC cells. MAGEA6 expression is correlated with AMPKα1 downregulation in RCC tissues and cells. It is not detected in normal renal tissues nor in the HK-2 renal epithelial cells. MAGEA6 knockdown by targeted-shRNA induced AMPK stabilization and activation, which led to mTOR complex 1 (mTORC1) in-activation and RCC cell death/apoptosis. AMPK inhibition, by AMPKα1 shRNA or the dominant negative AMPKα1 (T172A), almost reversed MAGEA6 knockdown-induced RCC cell apoptosis. Conversely, expression of the constitutive-active AMPKα1 (T172D) mimicked the actions by MAGEA6 shRNA. In vivo, MAGEA6 shRNA-bearing 786-O tumors grew significantly slower in nude mice than the control tumors. AMPKα1 stabilization and activation as well as mTORC1 in-activation were detected in MAGEA6 shRNA tumor tissues.
MAGEA6 knockdown inhibits human RCC cells via activating AMPK signaling.
背景/目的：黑色素瘤抗原A6（MAGEA6）是一种癌症特异性的AMP激活蛋白激酶（AMPK）泛素连接酶。本研究检测了MAGEA6在肾细胞癌（RCC）中的表达及潜在功能。
采用蛋白质免疫印迹法和qRT-PCR法检测人RCC组织及RCC细胞中MAGEA6和AMPK的表达。应用shRNA方法敲低人RCC细胞中的MAGEA6。分别采用MTT法和BrdU ELISA法检测细胞存活和增殖情况。采用TUNEL法和单链DNA ELISA法检测细胞凋亡情况。建立786-O裸鼠异种移植模型以检测RCC细胞在体内的生长情况。
MAGEA6在RCC组织以及已建立的（786-O和A498）和原代人RCC细胞中特异性表达。MAGEA6的表达与RCC组织和细胞中AMPKα1的下调相关。在正常肾组织和HK-2肾上皮细胞中未检测到MAGEA6。靶向shRNA敲低MAGEA6可诱导AMPK稳定和激活，进而导致mTOR复合物1（mTORC1）失活和RCC细胞死亡/凋亡。通过AMPKα1 shRNA或显性负性AMPKα1（T172A）抑制AMPK，几乎可逆转MAGEA6敲低诱导的RCC细胞凋亡。相反，组成型活性AMPKα1（T172D）的表达模拟了MAGEA6 shRNA的作用。在体内，携带MAGEA6 shRNA的786-O肿瘤在裸鼠中的生长明显慢于对照肿瘤。在MAGEA6 shRNA肿瘤组织中检测到AMPKα1稳定和激活以及mTORC1失活。
敲低MAGEA6通过激活AMPK信号通路抑制人RCC细胞。
