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目的:通过构建携带针对miR-194的慢病毒沉默及过表达载体,研究miR-194对骨肉瘤细胞转移的影响。方法:以miR-194前体及成熟体序列为模板,设计引物,进行PCR扩增,酶切后克隆于慢病毒载体plent-i GFP中,转染293T细胞,包装慢病毒颗粒,检测滴度,最终对人骨肉瘤细胞系U2-OS进行感染。获取稳定感染细胞后,进行Transwell、划痕等实验。结果:1)慢病毒表达质粒构建成功,沉默表达及过表达重组慢病毒的滴度分别为4×108TU/mL及1.5×108TU/mL;2)稳定过表达miR-194的人骨肉瘤细胞系U2-OS的转移能力较其他组有明显降低(P<0.01)。结论:成功构建了miR-194慢病毒表达载体,miR-194的表达水平对U2-OS细胞的转移能力具有显著影响,miR-194可能成为骨肉瘤治疗的潜在新靶点。
OBJECTIVE: To study the effect of miR-194 on osteosarcoma cell metastasis by constructing a lentivirus-silenced and overexpression vector carrying miR-194. Methods: Primers were designed and used for PCR amplification with miR-194 precursor and mature sequence as templates. After digestion, the PCR products were cloned into lentiviral vector plent-i GFP and transfected into 293T cells. The lentivirus particles were packaged and the titer , Eventually infecting human osteosarcoma cell line U2-OS. After obtaining stable infected cells, Transwell, scratch and other experiments were performed. Results: 1) The lentiviral expression plasmid was successfully constructed. The titer of the recombinant lentivirus was 4 × 108TU / mL and 1.5 × 108TU / mL, respectively.2) The human osteosarcoma cell line U2 stably overexpressing miR-194 -OS transfer capacity than other groups were significantly reduced (P <0.01). Conclusion: miR-194 lentiviral vector was successfully constructed. The expression of miR-194 has a significant effect on the metastasis of U2-OS cells. MiR-194 may be a potential new target for the treatment of osteosarcoma.