miR-194慢病毒表达质粒的构建及对人骨肉瘤细胞系U2-OS转移的相关研究

摘要

Objective:This study aimed to construct a lentiviral expression vector for microRNA-194 and investigate its effect on the metastasis of human osteosarcoma cell line U2-OS. Methods:Pri-and mature miR-194 amplified by PCR were inserted into the plenty-GFP vector and identified by restriction endonuclease digestion and nucleotide sequencing. The osteosarcoma cell line U2-OS was transfected with the lentivirus. Then, the stable transfected cells were used in Transwell and wound healing assay. Results:Restric-tion analysis and sequencing showed that the recombinant lentiviral expression vector was constructed correctly. The titers of obtained overexpression and suppression expression recombinant lentivirus were 1.5*108 and 4*108 TU/ml. Cell metastasis ability was signifi-cantly different in different experimental groups (P<0.01). Conclusion:The lentiviral expression vector for microRNA-194 was suc-cessfully constructed. MicroRNA-194 could influence the metastasis of the osteosarcoma cell line U2-OS;thus, it could be further ex-plored as a potential target in osteosarcoma therapy.%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。