Lentiviral vectors carrying siRNA inhibit S1PR3 gene expression in the corpus cavernosum smooth muscle cells of rats with spontaneous hypertension.
- Author:
Bang-Cai WU
1
;
Ji-Yi XIA
1
;
Rui JIANG
1
;
Hai-Fan YANG
1
Author Information
1. Department of Urology, The Affiliated Hospital of Southwest Medical University, Luzhou, Sichuan 646000, China.
- Publication Type:Journal Article
- Keywords:
RNA interference;
corpus cavernosum smooth muscle cell;
lentiviral vector;
sphingosine-1-phosphate receptor 3;
spontaneous hypertension rat
- MeSH:
Animals;
Down-Regulation;
Gene Expression;
Genetic Vectors;
Green Fluorescent Proteins;
metabolism;
Lentivirus;
genetics;
Male;
Myocytes, Smooth Muscle;
metabolism;
Nitric Oxide Synthase Type III;
metabolism;
Penis;
metabolism;
RNA, Messenger;
RNA, Small Interfering;
genetics;
metabolism;
Random Allocation;
Rats;
Rats, Inbred WKY;
Receptors, Lysosphingolipid;
genetics;
metabolism;
Signal Transduction;
Sphingosine-1-Phosphate Receptors;
Transfection;
rho-Associated Kinases;
metabolism
- From:
National Journal of Andrology
2017;23(2):110-119
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To screen lentiviral vectors carrying siRNA which can specifically down-regulate the gene expression of the sphingosine-1-phosphate receptor 3 (S1PR3) in the corpus cavernosum smooth muscle (CCSM) cells of rats with spontaneous hypertension (SHT) and investigate the influence of the vectors on the signaling pathways of ROCK1, ROCK2 and eNOS in the CCSM cells of SHT rats.
METHODS:Using the S1PR3 mRNA sequence of the rat as an interfering target, we designed and synthesized three pairs of siRNA sequences (siRNA1, 2 and 3) targeting S1PR3 and one pair of negative control, and then constructed and packaged them into lentiviral vectors. We cultured the CCSM cells of SHT and Wistar-Kyoto (WKY) rats in vitro and randomly divided them into groups A (SHT untransfected control), B (SHT transfected and carrying negative control virus), C (SHT transfected and carrying siRNA1 targeting S1PR3), D (SHT transfected and carrying siRNA2 targeting S1PR3), E (SHT transfected and carrying siRNA3 targeting S1PR3), and F (WKY untransfected control). With the multiplicity of infection (MOI) = 60, we transfected the CCSM cells of the SHT rats with the lentiviral vector and then determined the expression of the green fluorescent protein (GFP) as well as the mRNA and protein expressions of S1PR3, ROCK1, ROCK2 and eNOS in the CCSM cells of the SHT and WKY rats by RT-PCR and Western blot.
RESULTS:Gene sequencing proved the successful construction of the lentiviral vector. The transfection efficiency of the CCSM cells of the rats was >80% in groups B, C, D and E. Compared with group A, the mRNA and protein expressions of S1PR3, ROCK1 and ROCK2 exhibited no significant difference in group B but were remarkably decreased in groups C, D, E and F (P< 0.05), most significantly in group E, with the inhibition rates of the mRNA and protein expressions of S1PR3 of (34.2±2.9) and (77.7±4.7)%, those of ROCK1 of (33.3±1.4) and (51.1±7.3)%, and those of ROCK2 of (30.8±3.6) and (58.32±5.5)%, respectively. The mRNA and protein expressions of eNOS in group A showed no significant difference from those in groups B, C, D and E (P>0.05) but remarkably lower than those in group F (P< 0.05). Compared with group F, the mRNA and protein expressions of S1PR3, ROCK1 and ROCK2 were not significantly different from those in group E (P>0.05) but markedly increased in groups A, B, C and D (P< 0.05), while those of eNOS remarkably decreased in groups A, B, C, D and E (P< 0.05).
CONCLUSIONS:The three constructed lentiviral vectors carrying siRNA targeting different loci of the S1PR3 gene could significantly inhibit the expression of S1P3 as well as RhoA/Rho kinase signaling pathways in the CCSM cells of SHT rats, and the vector carrying siRNA3 exhibited the highest inhibitory effect.
