Screening Lentiviral Vectors Carrying Effective SiRNA Of The S1P2 Gene

Objective: To screen the lentiviral vector carrying siRNA with higher efficiency of suppressing the sphingosine-1-phosphate receptor 2(S1P2)gene expression in the primary cultured corpus cavernosum smooth muscle cells of spontaneously hypertensive rats(SHR).Methods: SHR and SD rats(n=5 each)were used for primary cultivating corpus cavernosum smooth muscle cells.The cells were randomly divided into group A to group C(respectively carrying targeted to S1P2 gene siRNA 1 ~ 3 targets of lentivirus SHR transfection group),group D(SHR GFP lentiviral transfection),group E(SHR non transfection group),and group F(SD rats control group),each group had 5 samples with 1.0±?105 cells of each sample.At 72 h after transfection(MOI =60)into the SHR corpus cavernosum smooth muscle cells with siRNA targeting S1P2 fragment of lentiviral vectors,the expression of GFP was observed under the fluorescent microscope.The protein expression of S1P2,eNOS,ROCK1,ROCK2 and mRNA expression of the S1P2,ROCK1,ROCK2 gene were determined by by Western blotting and RT-PCR.Results: The transfection efficiency of the group A,group B,group C,group D corpus cavernosum smooth muscle cells were>80%.Compared with the mRNA,protein expression in group E [S1P2(0.874 ± 0.047),(0.690 ± 0.122);ROCK1(0.819 ± 0.055),(0.731 ± 0.134);ROCK2(0.854 ± 0.098),(0.852 ±0.128)],group D [S1P2(0.905 ± 0.091),(0.701 ± 0.155);ROCK1(0.871 ± 0.073),(0.774 ± 0.135);ROCK2(0.857 ± 0.131),(0.825 ± 0.115)] showed no remarkablechanges(P> 0.05),group A [S1P2(0.560 ± 0.169),(0.238 ± 0.027);ROCK1(0.630 ± 0.059),(0.421 ± 0.160);ROCK2(0.614 ± 0.127),(0.486 ± 0.189)],group B [S1P2(0.731 ± 0.126),(0.501 ± 0.056),ROCK1(0.726 ± 0.057),(0.567 ±0.134),ROCK2(0.724 ± 0.146),(0.652 ± 0.119)],group C [S1P2(0.624 ±0.008),(0.431 ± 0.083);ROCK1(0.702 ± 0.051),(0.568 ± 0.117);ROCK2(0.649± 0.166),(0.600 ± 0.102)],group F [S1P2(0.540 ± 0.167),(0.147 ± 0.046);ROCK1(0.511 ± 0.152),(0.291 ± 0.107);ROCK2(0.568 ± 0.068),(0.474 ± 0.207)]was significantly lower than that in group D(P <0.05).The eNOS protein expression in group A(0.495 ± 0.156),group B(0.535 ± 0.098),group C(0.536 ±0.112),group D(0.551 ± 0.165)were not significant change compared with group E(0.518 ± 0.155),but the group F(0.888 ± 0.691)was significantly higher than group E.Conclusion: The expression of S1P2 gene in SHR corpus cavernosum smooth muscle cells was up-regulated and the RhoA / Rho kinase signaling pathway was activated.3 groups siRNA lentiviral vector targeting of S1P2 can inhibited the expression of S1P2 in corpus cavernosum smooth muscle of SHR,and by silencing S1P2 expression,reduced the expression of Rock1 and Rock2,Rho A / Rho kinase signaling pathway is inhibited,among them the inhibitory efficiency of the highest level is the siRNA1.