Effect Of Gene Silence With SiRNA Targeting To RhoA On Erectile Function Of Diabetic Rats

Part I Construction and amplification of eukaryotic expression vector coding shRNA targeting to rat RhoAmRNAObjectiveWe construct a recombinant plasmid vector that codes shRNA targeting to rat RhoAmRNA(pGPU6/RhoA-shRNA). The vector is designed for gene silence therapy of diabetic erectile dysfunction.MethodsThe RhoAmRNA-specific shRNA was designed according to rat RhoAmRNA sequence from the Genbank, and the sense and anti-sense strands of the specific shRNA template were artificially synthesized. The sense and anti-sense strands were annealled, and the double strands DNA were recombined with the pGPU6/GFP/Neo through enzyme digesting and ligation reactions. The product was transformed to E. Coli. DH5a, positive colonies were picked through kanamycin selection and plasmids were purified. The recombinant plasmids were further verified by restriction enzyme digestion and DNA sequencing. After the designed pGPU6/RhoA-shRNA was constructed successfully, the pGPU6/RhoA-shRNA was then amplified and purified for the later experiments.ResultsThe constructed pGPU6/RhoA-shRNA was digested by BamHI and EcoRI, and electrophoresed on1%agarose gel. the presented band was coincided with the size of formerly designed pGPU6/RhoA-shRNA. The recombinant plasmid identified by the restriction enzyme digestion was sent out for DNA sequencing. The sequencing result was completely identical with the formerly designed sequence.ConclusionThe eukaryotic expression vector coding shRNA targeting to rat RhoAmRNA (pGPU6/RhoA-shRNA) was successfully constructed with the techniques of molecular cloning. Part II modeling diabetic rat with erectile dysfunctionObjectiveWe model diabetic rats and determine the intracavernous pressures (ICP) of the rat, and verify that ICP of the diabetic rats are significant lower than that of healthy male rat at same age. The rat model is for the later sdudy on gene silence therapy of diabetic erectile dysfunction. MethodsTwenty eight Sprague-Dawley male rats, weighing220-250g, were randomly divided into experimental group(n=16) and control group(n=12). The rats of experimental group were intraperitoneally injected with streptozotocin(STZ) at60mg/kg, and the control group rats were injected with volume-mached solvent(0.1M PH4.5natrium citricum). After4d of the injection, the blood was collected from tail veins and blood glucose levels were measured. From then on, the blood glucose levels were measured for every two weeks. After10weeks of the injection, all the rats of the experimental group which were successfully-modeled to be diabetes mellitus(DM group) and all the control rats were determined for their basal intracavernous pressures(ICP) and neurostimulation-induced ICP. After the determination, all the rats were killed and their penises were harvested and the RhoAmRNA and RhoA protein expression in the penis tissue were determined.Results(DGeneral conditions In the experimental group,7wk and8wk after STZ injection, there respectively one rat died because of severe DM. There were one rat whose glucose level was under the creterion at8wk, and the rat was regarded to be resisted to STZ and was excluded from the successful DM models. At last,13of the16rats were succesfully modeded(regarded as DM group). The achievement ratio was81.3%.(2)Blood glucose and body weight4d、2wk、4wk、6wk、8wk and10wk After STZ injection, blood glucose levels of the DM group were significantly higher than those of the control goup respectively(P<0.001). There was no significant difference in the body weight between the DM and control groups before the STZ injection (P>0.05), but in the10th week after STZ injection, the body weight of DM group was significantly lower than that of control group(P<0.001), the mean body weight of the DM group was28.3%lower than that of the control group. (3)RhoAmRNA and RhoA protein expression In the10th week after STZ injection, the relative amounts of RhoAmRNA and RhoA protein in DM group were significantly higher than those in the control group respectively(P<0.001,P<0.002).(4)ICP In the10th week after STZ injection, the basal ICP and neurostimulation-induced ICP of DM group(10.6±3.6and46.5±16.3cmH2O) were significantly lower than those of control group(48.6±13.5cmH2O and140.1±33.9cmH2O)(all P<0.001). The basal ICP and neurostimulation-induced ICP decreased for76.4%and65.1%when compared with those of the control group.ConclusionIt is of high achievement ratio and stability to model DMED rats with STZ injection (60mg/kg, intraperitoneally), and it is of good relevance and sensitivity to measure the penile erectile function of rat through the basal ICP and neurostimulation-induced ICP. Part Ⅲ Effect of Gene Silence with SiRNA Targeting to RhoAmRNA on Erectile Function of Diabetic ratsObjectiveWe transfer SiRNA to the penile corpora cavernosa through pGPU6/RhoA-shRNA to specifically silence the RhoAmRNA, and investigate its effect on erectile function in DM rat with erectile dysfunction (DMED rat).Methods 102male DMED rats were randomly divided into three groups, group A treated with pGPU6/RhoA-shRNA, group B with pGPU6/NC, and group C with transferring reagent+PBS. At lwk,2wk,3wk and4wk after the treatment,8-9rats were randomly selected from each group and determined for their ICP, and then were killed and their penises were resected for testing RhoAmRNA and RhoA protein with RT-PCR and western-blot.Results(1)RhoAmRNA expression The relative amounts of RhoAmRNA in group A were significantly lowerer than those in group B and group C at lwk,2wk,3wk and4wk respectively(all P<0.001), but there were no significant differences in the amounts between group B and C(P>0.05). Within group A, B and C, there were no significant differences in the RhoAmRNA amounts among1wk,2wk,3wk and4wk(P>0.05).(2)RhoA protein expression The relative amounts of RhoA protein in group A were significantly lower than those in group B and group C at1wk,2wk,3wk and4wk respectively (all P<0.001), but there were no significant differences in the amounts between group B and C(P>0.05). Within group A, B and C, there were no significant differences in the RhoA protein amounts among1wk,2wk,3wk and4wk(P>0.05).(3)ICP①basal ICP There were no significant differences in the basal ICP among the three groups at lwk,2wk,3wk and4wk respectively(P>0.05). Within group A, B and C, there were no significant differences in the basal ICP among1wk,2wk,3wk and4wk respectively(P<0.05).②neurostimulation-induced ICP The neuro-stimulation-induced ICP of group A were significantly higher than those of group B and C at1wk,2wk,3wk and4wk respectively(P<0.001), but there were no significant differences in the neurostimulation-induced ICP between group B and C at1wk,2wk,3wk and4wk repectively(P>0.05). Within group A, B and C, there were no significant differences in the neurostimulation-induced ICP among1wk,2wk,3wk and4wk respectively (P<0.05).ConclusionThe pGPU6/RhoA-shRNA is successfully transferred to the penile corpora cavernosa in DMED rat, and it expresses RhoAmRNA and RhoA protein continuously and stably for at least4weeks. The gene silence targeting to RhoA can remarkably improve the erectile function of DMED rats.