建立慢病毒介导的miR-145稳定过表达的鼻咽癌细胞株

目的 构建过表达miR-145的慢病毒载体,建立稳定过表达miR-145的鼻咽癌细胞株.方法 构建慢病毒表达质粒pLVTHM/miR-145；293FT细胞进行慢病毒包装,生产的慢病毒感染鼻咽癌细胞株CNE1和CNE2,经流式细胞仪筛选后,建立稳定表达miR-145的鼻咽癌细胞株；最后应用荧光定量PCR检测病毒感染后鼻咽癌细胞株CNE1和CNE2中miR-145的表达水平.结果 荧光定量PCR结果和测序验证pLVTHM/miR-145重组质粒构建成功；包装生产的慢病毒感染鼻咽癌细胞CNE1和CNE2后,与阴性对照组比较,其表达水平分别升高4000倍和4500倍.结论 成功构建了pLVTHM/miR-145慢病毒重组质粒,建立稳定表达miR-145的鼻咽癌细胞株CNE1和CNE2,为研究miR-145在鼻咽癌发生发展过程中的基因调控和相应的作用机制提供了有用的细胞模型.

Establishment of A Nasopharyngeal Carcinoma Cell Lines Stably Over-Expressing miR-145 Mediated by Lentivirus

Objective To establish nasopharyngeal carcinoma （NPC） cell lines with stably over-expressing miR-145. Methods The recombinant lentivirus pLVTHM/miR-145 expression plasmid was packaged into mature lentivirus by 293gF cells and used to infect NPC cell lines CNE1 and CNE2. Flow cytometry was employed for sorting the GFP＋ cells. The efficiency of miR-145 over-expression was determined by qRT-PCR. Results The recombinant lentivirus plasmid pLVTHM/miR-145 was successfully constructed and verified by PCR and sequencing. The expression of miR-145 in CNE1 and CNE2 cells infected with the lentivirus pLVTHM/miR-145 were significantly up regulated as compared with the negative control groups. Conclusion CNE1 and CNE2 cells transfected with the recombinant lentivirus vector pLVTHM/ miR-145 showed high and stable expression of miR-145, which provide useful cell models for further studies of the role of miR-145 in nasopharyngeal carcinoma.