Gene Silencing By Systemic Delivery Of Adenovirus Vector-mediated Short Hairpin RNA Of Rat Angiotensin Receptor Subtype 1 In Spontaneously Hypertensive Rats

Part IConstruction and identification of eukaryoticexpression plasmid pGenesil-1-shRNA expressing theshRNA of rat angiotensin II receptorObjective: To construct the expression vector of shRNA targeted to the rat angiotensin II receptor gene for antihypertensive therapy in spontaneous hypertensive rat (SHR) at post-transcriptional level.Methods: In this study, the sense and antisense RNA oligonucleotides strands targeting angiotensin II receptor mRNA(Accession No. NM030985) were synthesized individually according to the sequence of the rat angiotensin II receptor. For preparation of duplexes, sense- and antisense-stranded oligonucleotides (20 pM each) were mixed together in annealing buffer (final concentration of 50 mM NaCl and Tris-HCL, pH8.0), incubating in 94°C for 1 min, and cooling slowly to roomtemperature, then the annealed duplexes were cloned into the BarnH I and Hind HI site of the pGenesil-1 vector, generating the construct pGenesil-1-shRNA.Results: The cloned shRNA construct which digested with Pst I and Sal I enzymes was inserted into the pGenesil-1 vector and verified with restriction enzymes , and the inserted sequences were verified by DNA sequencing, the plasmid pGenesil-1 -shRNA vector was gained.Conclusion: Plasmid-based shRNA expression systems targed against the rat angiotensin II receptor gene were generated successfully, the shRNAs with a 22-nt stem and a short loop were cleaved into small interfering dsRNA (siRNA) by the Dicer ,the siRNA enables the effective silencing of gene expression by presenting various siRNA sequence to the target mRNA and will be useful for the antihypertensive research in SHR.Part IIExpression in mammalian cells in vitro of the plasmid expressing shRNA targeted to the rat AT1 receptor geneExperiment 1Gene silencing in mammalian cells by the shRNA expression plasmids against the rat AT1 receptor geneObjective: To construct the expression vector of shRNA targeted to the rat angiotensin II receptor gene and to investigate the efficacy of preformed siRNAs in vitro to modulate the expression of target gene in mammalian cells for antihypertensive therapy in spontaneous hypertensive rats (SHR) at post-transcriptional level.Methods: In this study, 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 , then 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 phase. RT-PCR and western blot were performed.Results: The AT1 mRNA and protein levels behaved ultimately same. Compared to control after 48 hour, AT1 mRNA levels drop to 35.5 ± 3%, and the levels reach their lowest point after 72 hour (20.7±4% of control). At 24 hour and 48 hour, the AT1 protein was 46.9±4.2% and 36.98 ± 3.7% respectively compared to control and a maximum reduction was observed after 72 hour of incubation (28.1 ± 4% compared to controls).Conclusion: Plasmid-based shRNA expression systems targed against the rat angiotensin II receptor gene were generated successfully, the shRNAs 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 rat for the action, curative effect and application value and helpful to genetic therapy of the hypertension.Experiment 2Selection of effective shRNA sequences for gene silencing against rat angiotensin II subtype 1 receptorObjective: RNA interference (RNAi) is a recent technological advance thatenables researchers to reduce gene expression at the post-transcriptional level. A critical factor for effective siRNA is the choice of target sequence on the gene. Here, we have investigated the efficacy of preformed shRNAs to modulate the expression of rat angiotensin II subtype 1 receptor gene in mammalian cells and explored correlations of features of efficient shRNA with shRNA functionality.Methods: In this study, we generated four short hairpin RNA expression vectors targeting against rat angiotensin II receptor. The shRNAs were targeted to four different regions of same gene. The sense and antisense RNA oligonucleotides strands 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 , then 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 phase. RT-PCR and western blot were performed.Results: After 24 hour, no significant reduction in AT1 mRNA levels at all treated groups could be detected. Compared to control after 48 hour, AT1 mRNA levels of Pb treated group drop to 55.7±7.6%, and the levels reach their lowest point after 72 hour (43.7 ±8.2% of control). No significant inhibition of AT1 receptor mRNA expression was found in rat glioma cells transfected with other plasmids and scramble shRNA (P>0.05).The AT1 mRNA and protein levels behaved ultimately same. At 24 hour and 48 hour, the AT1 protein was 46.9±4.2% and 36.98 ± 3.7% respectively compared to control and a maximum reduction was observed after 72 hour of incubation (28.1 ±4% compared to controls).Conclustion: Plasmid-based shRNA expression systems targed against the rat angiotensin II receptor gene were generated successfully. The transcribed shRNA in transfected mammalian cells enables the effective silencing of gene expression to the target mRNA and leads to effective inhibition of translation of the proteins. 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.Part IIIConstruction of recombinant adenovirus expressing the short hairpin RNA of rat angiotensin II receptor subtype 1Objective : To construct the adenovirus vector expressing the shRNA of rat angiotensin II receptor and amplify the adenovirus vector in 293 cells.Methods: The rat AT1-shRNA segments was obtained from plasmid pGenesil-1-AT1R-shRNA which was constructed at an earlier date by RT-PCR and then inserted into linearized PUC18 plasmid. After having been screened, the constructed PUC18- AT1-shRNA plasmid was digested with restriction endonucleases and cloned into the shuttle plasmid PDC316 to form the PDC316- AT1-shRNA vector. The PDC316- AT1-shRNA plasmid was cotransfected with genomic plasmid pBHGlox Δ 1, 3Cre into 293 cells to package the recombinant adenovirus. The recombinant adenovirus was transfected into rat glioma C6 cells, and the green fluorescence protein expression was detected.Results: Recombinant adenoviral vector Ad5-AT1-shRNA was constructed successfully, which was confirmed by restriction enzyme digestion and PCR and GFP expression.Conclusion: The recombinant adenoviral vector carrying AT1-shRNA was successfully constructed, and will be lay the foundation of application of gene therapy to antihypertension.Part IVIn vivo study in SHR by Adenovirus vector-mediated shRNA targeted to the rat angiotensin II receptor subtype 1 geneExperiment 1 Effects on blood pressure in SHR by Adenovirus vector-mediatedshRNA targeted to the rat angiotensin II receptor subtype 1 geneObjective: To investigate the effects of adenoviral vector coding short hairpin RNA targeted to the rat angiotensin II receptor subtype 1 gene (Ad5-AT1R-shRNA) on blood pressure in spontaneously hypertensive rats (SHR), and the inhibitory effect of RNA interference (RNAi) on AT1R mRNA expression in SHR.Methods: SHRs (n=12) were randomly divided into two groups (n=6): SHR control group (SHR group) and experimental group. Six male Wistar-Kyoto rats(WKY) were selected as normal control group (WKY group). Ad5-ATlR-shRNA was constructed and propagated further in 293 cells. Systolic blood pressure (SBP) was measured before and after treatment with single intravenous injection of Ad5-ATlR-shRNA.The expression of AT1R mRNA was detected by quantitative RT-PCR. To identify the sites of Ad5-AT1R-shRNA expression, fluorescence microscope was used to observe different organs in the Ad5-ATlR-shRNA treated rats.Results: Administration of Ad5-AT1R-shRNA produced a prolonged duration of decrease in blood pressure. Furthermore, AT1R mRNA levels of kidney and aorta were decreased in the Ad5-ATlR-shRNA treated rats. Under the fluorescence microscope,we can see marked expression in heart, liver, kidney aorta and adrenal gland.Conclustion: The results indicate that Ad5-ATlR-shRNA can be absorbed by many main organs and inhibit the expression of AT1R mRNA to produce a prolonged duration of decrease in blood pressure. It is suggested RNA interference can be an effective approach of treating hypertension.Experiment 2Effects on left ventricular remodeling in SHR by Adcnovirus vector-mediated shRNA targeted to the rat angiotensin II receptor subtype 1 geneObjective: To investigate the effects of adenoviral vector expressing shRNA targeted to the rat angiotensin II receptor subtype 1 gene (Ad5-AT1R-shRNA) on ventricular remodeling in spontaneously hypertensive rats (SHR).Methods: SHRs (n=12) were randomly divided into two groups (n=6): SHR control group (SHR group) and experimental group. Six male Wistar-Kyoto rats(WKY) were selected as normal control group (WKY group).Systolic blood pressure (SBP) was measured before and after treatment with Ad5-ATlR-shRNA.Total heart weights (HW), body weights (BW) and heart weight-to-body weight ratios (HW/BW) were recorded. Myocardial hydroxyproline and collagen levels were measured. Myocardial ultrastructure was observed by transmission electron microscopy.Results: SBP in all SHR groups was much higher than in WKY group before experiment (P<0.01). SBP significantly decreased in all SHRs treated with Ad5-ATlR-shRNA (P<0.01) .No significant acute effect on SBP have been observed both in SHR group and WKY group after control vector injection (P>0.05). Compared with SHR control group, there was a significant descent in heart weight and HW/BW in experimental group (P<0.05, or P<0.01 ). The levels of myocardial hydroxyproline and collagen decreased merely in experimental group compared with SHR group (P<0.05), but myocardial hydroxyproline and collagen levels in SHR group were significantly higher than that in WKY group (P<0.05). After treatment of Ad5-AT1R-shRNA, the changes of myocardial ultrastructure in experimental group was significantly improved.Conclusion : Ad5-AT1R-shRNA improves significantly left ventricular remodeling and decreases blood pressure in SHR.Conclusion : Ad5-AT1R-shRNA improves significantly left ventricular remodeling and decreases blood pressure in SHR.