Department of Orthopedics, Xiangya Hospital, Central South University, Changsha, China.
Eur Rev Med Pharmacol Sci. 2019 Feb;23(3):982-991. doi: 10.26355/eurrev_201902_16985.
To study the effects of miR-340-5p on the proliferation of osteosarcoma (OS) U2OS cells.
miR-340-5p expression was measured by quantitative Real-time Polymerase Chain Reaction (qRT-PCR) in cancer tissues and paracancerous tissues from OS patients, and in OS cell line U2OS and normal osteoblast cell line hFOB1.19. The dual luciferase reporter vector was constructed to verify whether Signal transducers and activators of transcription 3 (STAT3) were a potential target gene of miR-340-5p. MiR-340-5p mimics/inhibitor was transfected into U2OS cells using liposomes, and transfection results were verified by qRT-PCR. Cell Counting Kit-8 (CCK-8) and Annexin V/PI were used to analyze the proliferation and apoptosis of transfected cells, respectively. Western Blot was used to detect STAT3 protein expression and Wnt/β-catenin pathway-related proteins after transfection. U2OS cells were injected into nude mice to observe the effects of over expression of miR-340-5p on tumor growth in vivo.
miR-340-5p gene expression in OS tissues and U2OS cells was significantly lower than that in paracancerous tissues and hFOB1.19 cells. miR-340-5p directly interacted with the 3'-untranslated region (3'-UTR) of STAT3 gene and negatively regulated its expression. The increase of miR-340-5p expression could significantly inhibit U2OS proliferation in vitro and induce apoptosis, and vice versa. STAT3, β-catenin, c-Myc, TCF-4, CyclinD1, and ROCK1 protein expression in U2OS cells was significantly decreased after miR-340-5p over-expression, and vice versa. MiR-340-5p over-expression in nude mice significantly decreased tumor size and weight.
Low expression of miR-340-5p in OS and U2OS cells could inhibit the course of OS by negatively regulating Wnt/β-catenin signaling pathway through targeting STAT3 gene. Therefore, miR-340-5p might be a potential biomarker and target for the diagnosis and treatment of OS.
研究 miR-340-5p 对骨肉瘤（OS）U2OS 细胞增殖的影响。
通过定量实时聚合酶链反应（qRT-PCR）测量 OS 患者癌组织和癌旁组织以及 OS 细胞系 U2OS 和正常成骨细胞系 hFOB1.19 中的 miR-340-5p 表达。构建双荧光素酶报告载体以验证信号转导和转录激活因子 3（STAT3）是否是 miR-340-5p 的潜在靶基因。使用脂质体将 miR-340-5p 模拟物/抑制剂转染 U2OS 细胞，并通过 qRT-PCR 验证转染结果。细胞计数试剂盒-8（CCK-8）和 Annexin V/PI 分别用于分析转染细胞的增殖和凋亡。Western Blot 用于检测转染后 STAT3 蛋白表达和 Wnt/β-catenin 通路相关蛋白。将 U2OS 细胞注射到裸鼠体内，观察 miR-340-5p 过表达对体内肿瘤生长的影响。
OS 组织和 U2OS 细胞中的 miR-340-5p 基因表达明显低于癌旁组织和 hFOB1.19 细胞。miR-340-5p 可直接与 STAT3 基因的 3'-非翻译区（3'-UTR）相互作用，并负调控其表达。体外 miR-340-5p 表达增加可显著抑制 U2OS 增殖并诱导细胞凋亡，反之亦然。miR-340-5p 过表达后 U2OS 细胞中 STAT3、β-catenin、c-Myc、TCF-4、CyclinD1 和 ROCK1 蛋白表达明显降低，反之亦然。裸鼠 miR-340-5p 过表达可显著降低肿瘤大小和重量。
OS 和 U2OS 细胞中 miR-340-5p 的低表达可通过负调控 STAT3 基因靶向抑制 Wnt/β-catenin 信号通路来抑制 OS 的发生。因此，miR-340-5p 可能是 OS 诊断和治疗的潜在生物标志物和靶点。
