Lentivirus Mediated SiRNA Interference Of S1PR3 Gene Expression In The SHR Corpus Cavernosum Smooth Muscl Cells

Objective:To screen the lentiviral vector carrying siRNA which can down-regulate specifically expression of S1PR3 gene in corpus cavernosum smooth muscle(CCSM)cells of spontaneously hypertensive rats(SHR)and research the impact of lentiviral vector selceted on the signal pathways of ROCK1,ROCK2 and eNOS in SHR CCSM cells.Methods:Taking S1P3 gene mRNA sequence of rats as a interfering target,we designed and synthesised three pairs of shRNA sequences(ShRNA1,ShRNA2,ShRNA3)targeting S1PR3 and one pair of negative control sequence,then built and packaged them into lentivirus vectors.We cultivated SHR and Wistar-Kyoto rats(WKY)CCSM cells in vitro,then divided them randomly SHR smooth muscle cells into five groups,that is group A(SHR CCSM cells),group B(Lentivirus transfection group without target sequence),C~E groups(Lentivirus transfection group with respectively siRNA targeting S1PR3 gene 1~3),group F(WKY CCSM cells).After 72 h since we transfected SHR CCSM cells with multiplicity of infection(MOI)=60,we observed the expression of green fluorescent protein(GFP)in cells with fluorescence microscope and tested the protein and mRNA expression of S1PR3,ROCK1,ROCK2 and eNOS in SHR and WKY CCSM cells with RT-PCR and Western blotting.Results:Gene sequencing proved that the sequence ofoligonucleotide sequences carrying S1PR3 gene siRNA 1~3 was inserted correctly.Under the fluorescence microscope,it was found that the siRNA lentiviral vector containing targeting S1PR3 and Negative control vector could effectively transfect the smooth muscle cells of SHR corpus cavernosum,the transfection efficiency was 80%.Compared with group A,the results of RT-PCR?Western blotting showed no significant change about mRNA and protein expression of S1PR3,ROCK1,ROCK2 in group B,but that in group C,D,E and F was decreased(p<0.05),among which inhibiting effect in group E was the most significant.The inhibition efficiency of mRNA and protein expression about S1PR3 was respectively(34.2±2.9)% ?(77.7±4.7)%,ROCK1 gene was respectively(33.3±1.4)% ?(51.1±7.3)% and ROCK2 gene was respectively(30.8±3.6)%?(58.32±5.5)%.Compared with group A,mRNA and protein expression of eNOS gene in group B,C and D showed no significant differences,but group F was significantly higher than group A(p<0.01).Compared with group F,we found the mRNA and protein expression of S1PR3,ROCK1,ROCK2 in group E showed no significant differences,but mRNA and protein expression of S1PR3,ROCK1,ROCK2 gene in groupA,B,C,D was higher than that in group F(p<0.05).mRNA and protein expression of eNOS gene in group A,B,C,D was decreased significantly than that in group F(p<0.01).Conclusion: In the study we built three siRNA targeting different loci of S1P3 gene and found these lentivirus vectors could significantly inhibit expression ofS1PR3 gene in SHR CCSM cells and could restrain effectively RhoA/Rho kinase signaling pathways in SHR CCSM cells.The ability of siRNA with different specific sequences to down-regulated expression of S1PR3,ROCK1 and ROCK2 gene varied,in which the third siRNA lentivirus vector revealed the strongest inhibitory effect because it could down-regulated the expression level of S1PR3,ROCK1 and ROCK2 in SHR CCSM cells and compared with WKY CCSM cells,there was no significant differences.