Construction of shRNA Targeted to the Rat Angiotensin Ⅱ Type 1 Receptors and Its RNAi in Cytoplasma

摘要

The expression vector of shRNA targeted to the rat angiotensin Ⅱ receptor gene was constructed and the efficacy of siRNAs to modulate the expression of target gene in the in vitro cultured mammalian cells was investigated for antihypertensive therapy in spontaneous hypertensive rat (SHR) at post-transcriptional level. The sense and antisense RNA oligonucleotides strands targeting angiotensin Ⅱ receptor mRNA were synthesized individually according to the sequence of the rat angiotensin Ⅱ receptor. For preparation of duplexes, sense- and antisense-stranded oligonucleotides were mixed and annealed, and the annealed duplexes were cloned into the pGenesil-1 vector. The rat glioma cells were transfected with constructed pGenesil-1-shRNA plasmid and scrambled plasmid. The cultured cells were collected at different phases. RT-PCR and Western blot were performed. The AT1 mRNA and protein levels behaved ultimately same. Compared to control after 48 h, AT1 mRNA levels were decreased to 35. 5 %±3. 0 %, and the levels reached their lowest point after 72 h (20. 7 %±4 % of control). At 24 and 48 h, AT1 protein was reduced to 46. 9 %±4. 2 % and 36. 98 % ±3. 7 % respectively compared to control and a maximum reduction was observed after 72 h of incubation (28. 1 % ±4 % compared to controls). Plasmid-based shRNA expression systems targeted against the rat angiotensin Ⅱ receptor gene were generated successfully. The shR-NAs with a 22-nt stem and a short loop were cleaved into small interfering dsRNA (siRNA) by the Dicer. The in vitro transcribed siRNA enables the effective silencing of gene expression to the target mRNA and leads to effective inhibition of translation of proteins and will be lay the foundation of application of gene silencing technology to hypertensive rats.