miR-296-5p 靶向 PLK1 调控 PI3K/ AKT 通路诱导骨肉瘤细胞中的自噬并抑制上皮-间质转化

目的 探讨 miR-296-5p 靶向 PLK1 对骨肉瘤 (osteosarcoma, OS) 细胞自噬及抑制上皮-间质转化(EMT) 的作用机制。 方法 qRT-PCR 检测 miR-296-5p 在 OS 细胞中的表达。 采用生物信息学分析预测miR-296-5p 的靶基因, 验证 miR-296-5p 对靶基因 PLK1 的直接靶向调控; 细胞转染构建 miR-296-5p 过表达和干扰细胞, CCK-8、 克隆形成、 Transwell 小室、 流式、 蛋白免疫印迹实验检测 miR-296-5p 的不同表达对U2OS 细胞中 PTBP1 表达水平及细胞增殖、 侵袭、 凋亡、 自噬及 EMT 的影响。 结果 与对照组比较, miR-296-5p 在 OS 中表达降低, 而 PLK1 则升高 (P< 0. 05); 与 miR-NC 组比较, mimic 组的克隆形成率、 侵袭细胞数目及 PTBP1、 p62、 N-cadherin、 Vimentin、 p-PI3K/ PI3K、 p-AKT/ AKT 水平降低, 细胞凋亡率、 Beclin-1、 LC3-Ⅱ/ Ⅰ、 E-cadherin 水平升高 (P< 0. 05); 与 PLK1 组比较, PLK1 + mimic 组的克隆形成率、 侵袭细胞数目及 PTBP1、 p62、 N-cadherin、 Vimentin、 p-PI3K/ PI3K、 p-AKT/ AKT 水平降低, 细胞凋亡率、 Beclin-1、 LC3-Ⅱ/ Ⅰ、 E-cadherin 水平升高 (P< 0. 05)。 结论 miR-296-5p 可能能够靶向 PLK1 调控 PI3K/ AKT通路诱导 OS 细胞中的自噬并抑制 EMT。

miR-296-5p Induces Autophagy in Osteosarcoma Cells and InhibitsEpithelial-mesenchymal Transition through Regulation of PI3K / AKTPathways by Targeting PLK1

Objective To explore the mechanism of miR-296-5p induced autophagy and inhibition of epithelial-mesenchymal transition ( EMT) in osteosarcoma (OS) cells. Methods Theexpression level of miR-296-5p in OS cells was detected by qRT-PCR. The target gene of miR-296-5p was predicted by bioinformatic method. The direct binding relationship of miR-296-5p with its target gene PLK1 was verified. Cell lines overexpressed or silenced of miR-296-5p were constructedthrough cell transfection method. The effect of miR-296-5p on cell proliferation, invasion, apoptosis, autophagy, EMT, and the expression level of PTBP1 in U2OS cells were detected by CCK-8,colony formation assay, transwell chamber assay, flow cytometry, and Western blotting. Results The expression level of miR-296-5p was decreased in OS, while the expression level of PLK1 wasincreased when compared to the control group (P < 0. 05). The colony formation rate, the numberof invasive cells, and the expression levels of PTBP1, p62, N-cadherin, Vimentin, p-PI3K/PI3K and p-AKT / AKT were decreased in mimic group, while the apoptosis rate, the expressionlevels of Beclin-1, LC3-Ⅱ/ Ⅰ and E-cadherin were increased when compared to the miR-NC group(P< 0. 05). The colony formation rate, the number of invasive cells, the expression levels of PTBP1, p62, N-cadherin, Vimentin, p-PI3K/ PI3K and p-AKT / AKT were decreased in PLK1 +mimic group, while the apoptosis rate, the expression levels of Beclin-1, LC3-Ⅱ/ Ⅰ and E-cadherin were increased in PLK1 + mimic group when compared to the PLK1 group (P< 0. 05). Conclusion miR-296-5p may induce autophagy in OS cells and inhibit EMT through regulation ofPI3K/ AKT pathways by targeting PLK1.