人细胞间黏附分子1基因慢病毒干扰载体感染MCF-7细胞下调靶基因表达

Down-regulation of human intercellular adhesion molecule-1 expression in MCF-7 cells infected by lentiviral short hairpin RNA interference vectors

目的构建人细胞间黏附分子1(ICAM-1)慢病毒干扰载体,包装病毒后,感染人乳腺癌细胞MCF-7并检测干扰效果。方法针对人ICAM-1基因设计并合成3条靶向短发夹RNA(shRNA)干扰序列(ICAM-1 shRNA1、ICAM-1 shRNA2和ICAM-1 shRNA3)以及一个阴性对照序列(NS),将其克隆入载体p LKO.1-SP6-PGK-GFP中,测序正确后利用3个质粒病毒包装系统(干扰质粒载体-ps PAX2-p MD2.G)转染HEK293T细胞并包装慢病毒。收获病毒上清并测定病毒滴度。将所获病毒感染MCF-7细胞,实时荧光定量PCR(qRT-PCR)和Western blot法检测干扰效率。结果菌落PCR结果显示目的片段成功插入p LKO.1-SP6-PGK-GFP载体中,DNA测序证实无误。将所制备之慢病毒感染MCF-7细胞,qRT-PCR和Western blot结果证实ICAM-1 shRNA3组ICAM-1 mRNA和蛋白水平有明显下降。结论成功构建了人ICAM-1基因shRNA慢病毒干扰载体,包装慢病毒后,感染MCF-7细胞可引起ICAM-1表达下调。

Objective To construct lentiviral interference vectors of human intercellular adhesion molecule-1（ICAM-1）,then infect human breast cancer MCF-7 cells and identify the interference effects. Methods Three short hairpin RNA（shRNA） interference sequences targeting human ICAM-1 gene（ICAM-1 shRNA1,ICAM-1 shRNA2 and ICAM-1 shRNA3）and a negative control sequence（ NS） were designed,synthesized and cloned into the p LKO. 1-SP6-PGK-GFP vector. After DNA sequencing,three plasmid-based lentiviral packaging system（ vector plasmid-ps PAX2-p MD2. G） was used to transfect HEK293 T cells to package lentiviruses. The supernatants containing viruses were harvested to detect the viral titer. Human MCF-7 breast cancer cells were infected with the lentiviruses and the interference efficiency was detected by real-time quantitative PCR（ qRT-PCR） and Western blotting. Results PCR showed that the designed sequences were successfully inserted into the p LKO. 1-SP6-PGK-GFP vector and DNA sequencing results were correct. The qRT-PCR and Western blotting showed that the mRNA and protein expression levels of ICAM-1 in the infected MCF-7 cells decreased significantly in the ICAM-1 shRNA3 group. Conclusion Lentiviral interference vectors of human ICAM-1 were constructed successfully and the expression of ICAM-1 in MCF-7 cells was down-regulated by ICAM-1 shRNA.