Gene silencing targeted to the rat AT1 receptors by efficient shRNAs and its screening

    QIU Ling1, XIAO Chuanª²Shi2,ZENG Qiuª²Tang1,LI Maoª²Lian2

    1Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China£¬ 2Second Hospital, Shanxi Medical University, Taiyuan 030001£¬ China

    ¡¾Abstract¡¿ AIM: To investigate the efficacy of short hairpin RNAs (shRNAs) to modulate the expression of rat angiotensin ¢ò typeª²1 (AT1) receptor gene in mammalian cells and to explore the correlations between the features of efficient shRNAs and shRNAs functionality. METHODS: We generated 4 shRNAs expression vectors targeting against rat angiotensin¢ò receptors. The shRNAs were targeted to 4 different regions of the same gene. The rat C6 glioma cells were transfected with constructed pGenesilª²1ª²shRNA plasmid and scrambled plasmid. The cultured cells were collected at different phases for RTª²PCR and Western blot analyses. RESULTS: 24 h later, no significant reduction in AT1 mRNA levels at all treated groups could be detected. 48 h later, AT1 mRNA level of Pb treated group droped to (55.7¡À7.6)% of that in control group, and reached their lowest point after 72 h (43.7¡À8.2)%. No significant inhibition of AT1 receptor mRNA expression was found in other groups (P>0.05). The AT1 mRNA and protein levels behaved ultimately similarly. At hour 24 and 48, the AT1 protein was (46.9¡À4.2)% and (37.0¡À3.7)% respectively compared to control and a maximum reduction was observed after 72 h incubation [(28.1¡À4.0% compared to controls)]. CONCLUSION: Besides some general rules, we believe that the loop structure in the mRNA at the region targeted by siRNA and the better accessibility of the siRNA to targeted mRNA do have a strong effect on silencing. ......