Pan Bao-Long, Tong Zong-Wu, Wu Ling, Pan Li, Li Jun-E, Huang You-Guang, Li Shu-De, Du Shi-Xun, Li Xu-Dong
Department of Laboratory, People's Hospital of Yuxi City, Yuxi, China.
Department of nephrology, People's Hospital of Yuxi City, Yuxi, China.
Cell Physiol Biochem. 2018;45(4):1410-1422. doi: 10.1159/000487567. Epub 2018 Feb 15.
BACKGROUND/AIMS: This study aimed to investigate the mechanism by which microRNA-206 (miR-206) affects the proliferation, apoptosis, migration and invasion of osteosarcoma (OS) cells by targeting ANXA2 via the AKT signaling pathway.
A total of 132 OS tissues and 120 osteochondroma tissues were examined in this study. The targeting relationship between miR-206 and ANXA2 was verified with a dual-luciferase reporter assay. The miR-206 expression and ANXA2, AKT, PARP, FASN, Survivin, Bax, Mcl-1 and Bcl-1 mRNA and protein expression in the above two groups were examined by qRT-PCR and western blotting. The cultured OS cells were divided into 6 groups: a blank group, negative control (NC) group, miR-206 mimic group, miR-206 inhibitor group, si-ANXA2 group and miR-206 inhibitor + si-ANXA2 group. Cell cycle and apoptosis were assessed by flow cytometry, cell migration was examined with a wound-healing assay, and cell invasion was assessed with a Transwell assay. Pearson correlation analysis was used to determine the correlation between ANXA2 mRNA expression and miR-206 expression in OS.
OS tissues exhibited increased mRNA and protein expression of ANXA2, AKT, PARP, FASN, Survivin, Mcl-1 and Bcl-2; decreased miR-206 expression; and decreased Bax mRNA and protein expression. ANXA2 mRNA expression was strongly negatively correlated with miR-206 expression in OS. ANXA2 was found to be a miR-206 target gene. In the miR-206 mimic group and the si-ANXA2 group, the mRNA and protein expression of ANXA2, AKT, PARP, FASN, Survivin, Mcl-1 and Bcl-1 decreased markedly, cell proliferation was inhibited, apoptosis was promoted, higher cell growth in G1 phase and decreased growth in S phase was detected, and decreased cell migration and invasion were observed compared with those in the blank group.
The current results demonstrate that miR-206 overexpression inhibits OS cell proliferation, migration and invasion and promotes apoptosis through targeting ANXA2 by blocking the AKT signaling pathway.
背景/目的：本研究旨在探究微小RNA-206（miR-206）通过AKT信号通路靶向膜联蛋白A2（ANXA2）影响骨肉瘤（OS）细胞增殖、凋亡、迁移和侵袭的机制。
本研究共检测了132例OS组织和120例骨软骨瘤组织。采用双荧光素酶报告基因检测法验证miR-206与ANXA2之间的靶向关系。通过qRT-PCR和蛋白质免疫印迹法检测上述两组中miR-206表达以及ANXA2、AKT、PARP、脂肪酸合酶（FASN）、生存素（Survivin）、 Bax、髓细胞白血病-1（Mcl-1）和Bcl-1的mRNA及蛋白表达。将培养的OS细胞分为6组：空白组、阴性对照组（NC）、miR-206模拟物组、miR-206抑制剂组、si-ANXA2组和miR-206抑制剂+si-ANXA2组。通过流式细胞术评估细胞周期和凋亡，采用伤口愈合试验检测细胞迁移，采用Transwell试验评估细胞侵袭。采用Pearson相关分析确定OS中ANXA2 mRNA表达与miR-206表达之间的相关性。
OS组织中ANXA2、AKT、PARP、FASN、Survivin、Mcl-1和Bcl-2的mRNA及蛋白表达增加；miR-206表达降低；Bax mRNA及蛋白表达降低。OS中ANXA2 mRNA表达与miR-206表达呈显著负相关。发现ANXA2是miR-206的靶基因。与空白组相比，在miR-206模拟物组和si-ANXA2组中，ANXA2、AKT、PARP、FASN、Survivin、Mcl-1和Bcl-1的mRNA及蛋白表达显著降低，细胞增殖受到抑制，凋亡得到促进，检测到G1期细胞生长增加而S期生长减少，并且细胞迁移和侵袭减少。
目前的结果表明，miR-206过表达通过阻断AKT信号通路靶向ANXA2，从而抑制OS细胞增殖、迁移和侵袭并促进凋亡。
