| 韩康 赵廷宝 卞娜 蔡成魁 颜世举 王鑫 肖春 沙浩 董川 杨彤涛 周勇 马保安.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[J].,2014,14(23):4401-4405 |
| 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 |
| Effects of MicroRNA-194 on Proliferation and Apoptosis ofHuman Osteosarcoma Cell Line U2-OS by UsingRecombinant Lentivirus Vector |
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| DOI： |
| 中文关键词: microRNA-194 慢病毒 骨肉瘤 细胞增殖 细胞凋亡 |
| 英文关键词: MicroRNA-194 Lentivirus Osteosarcoma Cell proliferation Cell apoptosis |
| 基金项目:国家自然科学基金项目（81201633） |
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| 中文摘要: |
| 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× 108 TU/ml及4× 108 TU/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。 |
| 英文摘要: |
| 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 (P<0.01).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. |
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