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SOSP-9607,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:构建沉默及过表达microRNA-194的慢病毒载体;获得稳定感染的细胞株,改变细胞增殖相关生物学特性。结果:PCR及测序结果证实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;microRNA-194沉默的人骨肉瘤细胞系SOSP-9607细胞增殖速度相较于其对照组明显上升;过表达microRNA-194的SOSP-9607细胞增殖速度显著下降(P<0.01)。MicroRNA-194沉默的人骨肉瘤细胞系SOSP-9607细胞凋亡率相较于其对照组明显下降,过表达microRNA-194的SOSP-9607细胞凋亡率显著上升(P<0.01)。与对照组比较,过表达microRNA-194的SOSP-9607细胞周期G0/G1细胞比例显著增加,G2/M期细胞比例显著减少,S期细胞比例显著减少(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SOSP一9607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。
Objective：shRNA vector and exPression vector of microRNA - 194 were constructed to Package lenti-virus that could knock down or overexPress microRNA - 194. MicroRNA - 194 levels in osteosarcoma cell line SOSP- 9607 were changed through lentivirus infection. Then the changes of cell Proliferation rate and cell cycle were ob-served. All these would Provide the foundation of theory and exPeriment to exPlore microRNA - 194 as a new target of osteosarcoma theraPy. Methods：To construct lentivirus Production infection and changes cell biological characteris-tics. Results：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 × 108 TU/ ml and 4 × 108 TU/ ml. The cell Proliferation rate of SOSP - 9607 - microRNA - 194 RNAi was higher than SOSP - 9607- microRNA - 194 CON,the cell Proliferation rate of SOSP - 9607 - microRNA - 194 was lower than SOSP - 9607- microRNA - 194 CON. The aPoPtosis ratio of SOSP - 9607 - microRNA - 194 RNAi was lower than SOSP - 9607- microRNA - 194 CON,the aPoPtosis rate of SOSP - 9607 - microRNA - 194 was higher than SOSP - 9607 - mi-croRNA - 194 CON. eas - miR - 194 increased the cell ratio of G0 / G1 Phase,simultaneously decreased significantly the cell ratio of G2 / M Phase and S Phase（P ﹤ 0. 01）. Conclusion：The lentiviral exPression vector for microRNA -194 was successfully constructed. microRNA - 194 could influence osteosarcoma cell line SOSP - 9607 Proliferation rate,aPoPtosis ratio and cell cycle. MicroRNA - 194 could be further exPlored as a Potential target in osteosarcoma theraPy.
出处
《现代肿瘤医学》
CAS
2014年第5期985-989,共5页
Journal of Modern Oncology
基金
国家自然科学基金资助项目(编号:81201633)
