利用慢病毒载体观察microRNA-194对人骨肉瘤细胞系U2-OS生物学特性的影响

目的：通过构建携带针对microRNA-194的shRNA载体及过表达载体,包装慢病毒,感染人骨肉瘤细胞系U2-OS,改变细胞内microRNA-194基因的表达水平,研究microRNA-194对细胞增殖、凋亡及细胞周期的影响,为探索microRNA-194作为骨肉瘤生物治疗的新靶点提供理论和实验依据。方法：PCR扩增pri-miR-194及miR-194-5成熟体,克隆于慢病毒载体plent-i GFP中,转染大肠杆菌感受态细胞,收获并浓缩慢病毒颗粒,感染人骨肉瘤细胞系U2-OS。进行MTT,流式,平板克隆等试验。结果：1.重组慢病毒表达质粒构建正确,过表达及抑制过表达重组慢病毒的滴度分别为1.5×108TU/ml及4×108TU/ml；2.microRNA-194过表达的人骨肉瘤细胞系U2-OS细胞增殖速度,凋亡率及细胞周期相较于其它实验组都有明显变化（P〈0.01）。结论：成功构建了microRNA-194过表达及抑制过表达慢病毒表达载体；miR-194的表达水平对U2-OS细胞的增殖和凋亡造成显著影响。microRNA-194可能成为骨肉瘤治疗的潜在新靶点,但该基因对骨肉瘤发生发展的作用及其机制尚待进一步研究。

Effects of MicroRNA-194 on Proliferation and Apoptosis of Human Osteosarcoma Cell Line U2-OS by Using Recombinant Lentivirus Vector

Objective： shRNA vector and expression vector of microRNA-194 were constructed to package lentivirus that could knock down or overexpress microRNA-194. MicroRNA-194 levels in Osteosarcoma Cell Line U2-OS were changed through lentivirus infection. Then the changes of cell proliferation rate and cell cycle were observed. All these would provide the foundation of theory and experiment to explore microRNA-194 as a new target of osteosarcoma therapy. Methods： Pri-miR-194 and mature miR-194-5 amplified by PCR was inserted into plenty-GFP vector, and then identified by restriction endonuclease digestion and nucleotide sequencing.Osteosarcoma Cell Line U2-OS was transfected with the Lentivirus. Then the cells were used in MTT, flow cytometry analysis and clone formation. Results： 1.Restriction analysis and sequencing proved that recombinant lentiviral expression vector was constructed correctly.The titer of obtained overexpression and suppression expression recombinant lentivirus was 1.5 ×108TU/ml and 4×108TU/ml. 2. The cell proliferation rate, the apoptosis ratio and the cell ratio of G0/G1 phase of U2-OS were obviously different in different experimental group（P0.01）. Conclusion： The lentiviral expression vector for microRNA-194 was successfully constructed. microRNA-194 could influence Osteosarcoma Cell Line U2-OS proliferation rate, apoptosis ratio and cell cycle. microRNA-194 could be further explored as a potential target in Osteosarcoma therapy.