人 microRNA -194过表达及抑制表达慢病毒载体的构建及对人骨肉瘤细胞系SOSP-9607生物学特性的影响

摘要

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.%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1.5×108 TU/ ml 及4×108 TU/ 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。