The Exploration Of The Treatment To ED By Knocking Down The Expression Of PDE5 In Smooth Muscle Cells Of Human Corpus Cavernosum Through SiRNA

Objective: To construct specific siRNA and to study the inhibition of phosphodiesterase-5 (PDE5) gene expression of human and rat with plasmid and adenovirus vector.Methods: According to the Homo sapiens PDE5A3 gene mRNA sequence, four strands of oligonucleotide were designed and chemically synthesized. The two DNA strands were ligated with plasmid vector respectively and then were transformed into E.coli DH5α. After selective culture, recombinated plasmids were extracted. Fluorescence was observed with the fluorescence microscope and the efficiency of transfection was determined by the flow cytometry after these plasmids were transfected into smooth muscle cells of human corpus cavernosum. The expression cassettes of three specific effective siRNA and enhanced green fluorescent protein (EGFP) were cloned in eukaryotic expression vector pGenesil-1 and then subcloned into the shuttle plasmid pShuttle2. These objective expressed cassettes were subcloned into backbone vector pAdeno-X and plasmids of recombinant adenovirus expressing three siRNA and EGFP were generated. These plasmids were linearized by restriction enzyme PacI after being confirmed by endonuclease and PCR and then were transformed into HEK293A. Further the recombinant adenovirus rAd5-siRNA-PDE5A3 was packaged and propagated in 293A cells. The fluorescence microscopy was employed to identify the packaged progress and efficiency of infection. Recombinant virus was detected by PCR with specific primer of siRNA. The titer of virus was determined by median tissue culture infective dose (TCID50). After the recommbinate virus were transfected to the CCMC of primary culture in vitro for 48h and 72h, the RT-PCR and Western blot methods are used for detecting the expression level of PDE5A3 gene in the CCMC. PDE5 gene was cloned from the primary cultured rat cavernous smooth muscle cells (CSMCs) via RT-PCR. The sequences of six small interfering RNAs (siRNAs) targeting PDE5 gene were designed, synthesized and subcloned into pGenesil-1 expression vector respectively to construct six recombinant plasmids. PDE5 expression cassette was further subcloned into pIRES2-EGFP, which bears an EGFP coding CDS, and was named PIRES2-EGFP-PDE5. 48 hours after cotransfecting siRNAs plasmid and PIRES2-EGFP-PDE5 into HEK293A cells, fluorescence was observed and the inhibition efficiency of the expression of rat PDE5 was detected by RT-PCR and Western blot. The expression cassettes of two specific effective siRNA and EGFP were cloned in eukaryotic expression vector pGenesil-1 and then subcloned into the shuttle plasmid pShuttle2. These objective expressed cassettes were subcloned into backbone vector pAdeno-X and plasmids of recombinant adenovirus expressing two siRNA and EGFP were generated. These plasmids were linearized by restriction enzyme PacI after being confirmed by endonuclease and PCR and then were transformed into HEK293A. Further the recombinant adenovirus rAd-siRNA-rPDE5 was packaged and propagated in 293A cells. The fluorescence microscopy was employed to identify the packaged progress and efficiency of infection. Recombinant virus was detected by PCR with specific primer of siRNA. The titer of virus was determined by median tissue culture infective dose (TCID50).Results: siRNAs and recombinant plasmid vector were identified by digestion with EcoRI and Hind III and confirmed by sequencing analysis. Bright green fluorescence was observed after transfection with the eukaryotic expression vector marked by EGFP on 48 hours. The average efficiency of transfection was 17.95%. Restriction enzyme digestion analysis and observed fluorescence and the sequence analysis by PCR confirmed that the recombinant adenovirus rAd5-siRNA-PDE5A3 containing three specific siRNA was successfully constructed. The titer of the recombinant adenovirus was 8×108 pfu/ml. EGFP can be observed in all 293A cells. The recombinant adenovirus rAd5-siRNA-PDE5A3 and control virusAd5-EGFP were amplified and purified successfully, thus, the 8.0×108 pfu/ml tite recombinant adenovirus and 6.5×108pfu/ml tite control viruswere obtained. The efficiency to transfect the CCMC was up to over 95%, as for PDE5A3 gene expressed on mRNA level inhibition, it is (71.7±2.30)%as 48h,it is (70.8±3.61)%as 72h,as for protein level inhibition, it is (78.3±3.33)%as 48h,it is (77.5±2.67)% (P<0.01) as 72h. siRNAs for rat'PDE5 and PIRES2-EGFP-PDE5 were identified by endo-nuclease digestion and confirmed by sequencing. Green fluorescence observation showed that the EGFP level in cells which cotransfected with specific siRNA expression plasmids obviously weaker than control group. Further examination through RT-PCR and Western blot indicated that the highest inhibition ratio of PDE5 mRNA and protein by Pgenesil-2-siRNA4 was up to (68.59±3.23)%* and (71.25±2.91)* (*P<0.01) respectively, and the second significant one by Pgenesil-2-siRNA6 reached (55.02±3.44)* and (65.09±3.05)*(*P<0.01).Restriction enzyme digestion analysis and observed fluorescence and the sequence analysis by PCR confirmed that the recombinant adenovirus rAd-siRNA-rPDE5 containing two specific siRNA was successfully constructed. The titer of the recombinant adenovirus was 8×108 pfu/ml. EGFP can be observed in all 293A cells.Conclusion: Eukaryotic expression vector of siRNA specific for PDE5A3 gene has been constructed successfully. The shRNAs expressed by adenoviral vector can inhibit obviously the expression of CCMC PDE5A3 gene, which will open a way for further treatment and research of erectile dysfunction. The expression of rat PDE5 gene in HEK293A could be significantly decreased by RNA interference. The recombinant adenovirus rAd-siRNA-rPDE5 expressing efficiently two pairs of specific siRNA can be used further in gene therapy of erectile dysfunction.