shRNAmir慢病毒载体沉默鼻咽癌细胞中环氧合酶-2的表达

目的 构建基于miR-155结构的shRNAmir慢病毒表达载体,在鼻咽癌细胞中沉默COX-2基因的表达,探索全新的COX-2抑制方法.方法 转染小干扰RNA(siRNA),筛选出沉默COX-2表达的RNA干扰序列,以该序列替换miR-155前体序列中的双链部分作为anti-COX-2 shRNAmir转录模板,构建pLVTHM/shRNAmir慢病毒质粒表达载体,利用293FT细胞包装pLVTHM/anti-COX-2 shRNAmir慢病毒.感染鼻咽癌C666-1细胞后,通过流式分选,建立anti-COX-2 shRNAmir稳定表达,COX-2基因沉默的亚系.结果 设计合成的anti-COX-2a siRNA能够使COX-2 mRNA表达下降90%以上,根据其序列构建的plVTHM/sbRNLAmir质粒载体测序无误,pLVTHM/anti-COX-2 shRNAmir慢病毒感染C666-1细胞后,分选出亚系C666-1经反复传代仍可保持COX-2基因沉默的表型.结论 pLVTHM/shRNAmir慢病毒载体能够转录出基于miR-155结构的shRNAmir,沉默鼻咽癌细胞COX-2的表达,为探索更加高效安全的COX-2抑制剂奠定基础,也为鼻咽癌研究和抗肿瘤基因治疗提供了新手段.

Silencing of COX-2 in nasopharyngeal carcinoma cells with a shRNAmir lentivirus vector

Objective To construct a miR-155-based lentivirus vector to induce cyclooxygenase-2 gene silencing in nasopharyngeal carcinoma (NPC) cells by expressing anti-COX-2 shRNAmir. Methods miR-155-based anti-COX-2 shRNAmir template was synthesized and inserted into pLVTHM plasmid. The recombinant pLVTHM/shRNAmir was transfected into 293FT cells for packaging the lentivirus vector. After infection with the lentivirus vector, the GFP-positive cells were screened by flow cytometry, and COX-2 mRNA level was detected b...