Cao Riliang, Shao Jianli, Hu Yabin, Wang Liang, Li Zhizhong, Sun Guodong, Gao Xiaoliang
Department of Pediatric Surgery, Guangdong Women and Children Hospital, Guangzhou, 511400, China.
Department of Orthopedic and Traumatology, First Affiliated Hospital, Jinan University, Guangzhou, 510632, China.
Cancer Cell Int. 2018 Apr 2;18:49. doi: 10.1186/s12935-018-0551-x. eCollection 2018.
Osteosarcoma (OS) is a rare, malignant bone tumor that primarily affects adolescents and has a high degree of malignancy and high incidence of recurrence and metastasis. Our study aimed to explore the role of miR-338-3p in OS cells.
qRT-qPCR was performed to quantify miR-338-3p expression levels in OS tissue samples and in three common OS cell lines. MG-63 and Saos2 cells were separately transfected with miR-338-3p or NC mimics and miR-338-3p expression levels was determined by qRT-PCR. Cell proliferation was monitored using the Cell Counting Kit-8. Flow cytometer analysis was carried out to determine the distribution of cell cycle stages and apoptosis. Transwell assay was performed to measure the migratory and invasive capacities of MG-63 and Saos2 cells. The expression of Vimentin and E-cadherin was detected by western blot. Luciferase reporter assay, qRT-PCR and western blotting were performed to confirm the target of miR-338-3p.
Analysis by qRT-PCR revealed that miR-338-3p was downregulated in the tissue samples of 20 OS patients when compared with that in their matched adjacent non-tumor tissues. Furthermore, miR-338-3p was significantly downregulated in three common OS cell lines, namely, MG-63, Saos2, and HOS, when compared with that in the human osteoblast cell line hFOB1.19. Analysis by luciferase reporter assay, qRT-PCR, and western blotting revealed that activator of 90 kDa heat shock protein ATPase homolog 1 (AHSA1) is a direct target of miR-338-3p. miR-338-3p overexpression led to significant reduction in AHSA1 protein levels in MG63 and Saos2 cells. miR-338-3p overexpression reduced cell viability and migration and invasion behavior of MG63 and Saos2 cells. In addition, miR-338-3p overexpression suppressed epithelial-mesenchymal transition (EMT), induced a significant G1-phase arrest and did not affect the apoptosis in both MG-63 and Saos2 cells. Moreover, overexpression of AHSA1 reversed the inhibitory effect of miR-338-3p overexpression on proliferation, cell cycle, apoptosis, EMT, migration, and invasion of MG63 and Saos2 cells, thereby suggesting that miR-338-3p acts as a tumor suppressor in OS cells by targeting AHSA1.
miR-338-3p/AHSA1 can serve as a potential therapeutic target for OS therapy.
骨肉瘤（OS）是一种罕见的恶性骨肿瘤，主要影响青少年，具有高度恶性以及高复发和转移率。我们的研究旨在探讨miR-338-3p在骨肉瘤细胞中的作用。
采用qRT-qPCR法检测骨肉瘤组织样本及三种常见骨肉瘤细胞系中miR-338-3p的表达水平。分别用miR-338-3p或阴性对照模拟物转染MG-63和Saos2细胞，通过qRT-PCR检测miR-338-3p表达水平。使用细胞计数试剂盒-8监测细胞增殖。通过流式细胞仪分析确定细胞周期阶段分布和凋亡情况。进行Transwell实验以检测MG-63和Saos2细胞的迁移和侵袭能力。通过蛋白质免疫印迹法检测波形蛋白和E-钙黏蛋白的表达。进行荧光素酶报告基因检测、qRT-PCR和蛋白质免疫印迹以确认miR-338-3p的靶标。
qRT-PCR分析显示，与20例骨肉瘤患者配对的相邻非肿瘤组织相比，miR-338-3p在骨肉瘤组织样本中表达下调。此外，与人类成骨细胞系hFOB1.19相比，miR-338-3p在三种常见骨肉瘤细胞系MG-63、Saos2和HOS中显著下调。荧光素酶报告基因检测、qRT-PCR和蛋白质免疫印迹分析显示，90 kDa热休克蛋白ATP酶同源物1（AHSA1）是miR-338-3p的直接靶标。miR-338-3p过表达导致MG63和Saos2细胞中AHSA1蛋白水平显著降低。miR-338-3p过表达降低了MG63和Saos2细胞的活力以及迁移和侵袭能力。此外，miR-338-3p过表达抑制上皮-间质转化（EMT），诱导显著的G1期阻滞，且不影响MG-63和Saos2细胞的凋亡。此外，AHSA1过表达逆转了miR-338-3p过表达对MG63和Saos2细胞增殖、细胞周期、凋亡、EMT、迁移和侵袭的抑制作用，从而表明miR-338-3p通过靶向AHSA1在骨肉瘤细胞中发挥肿瘤抑制作用。
miR-338-3p/AHSA1可作为骨肉瘤治疗的潜在治疗靶点。
