The evaluation of efflux transporter model based on RNA interference technology in vitro.
- Author:
Ling-lei KONG
;
Hai-ying YANG
;
Mei YUAN
;
Xiao-mei ZHUANG
;
Hua LI
- Publication Type:Journal Article
- MeSH:
ATP-Binding Cassette, Sub-Family B, Member 1;
genetics;
Gene Knockdown Techniques;
Multidrug Resistance-Associated Proteins;
genetics;
RNA Interference;
RNA, Small Interfering;
Real-Time Polymerase Chain Reaction
- From:
Acta Pharmaceutica Sinica
2015;50(9):1122-1127
- CountryChina
- Language:Chinese
-
Abstract:
In the present study, the specifically knockdown models of P-gp or MRP2 were constructed by using a series of chemically synthesized small interfering RNA (siRNA) in vitro. The expression of P-gp and MRP2 was measured by real-time PCR and Western blot, and the function was evaluated by applying P-gp and MRP2 substrate, rhodamine and methotrexate. The results showed that MRP2 siRNA-3 or P-gp siRNA-2 significantly decreased the mRNA expression of MRP2 or P-gp, the inhibition ratio was 68% or 84%; MRP2 siRNA-3 or P-gp siRNA-2 at a dose of 80 nmol x L(-1) significantly reduced the protein expression of MRP2 or P-gp at 48 h after treatment, the inhibition ratio was 62% or 70%. Meanwhile, other transporters were not influenced by siRNA. When pretreatment with MRP2 siRNA-3 or P-gp siRNA-2, the efflux of methotrexate or rhodamine decreased significantly and the intra-cellular concentration increased. The results suggested that chemically synthesized siRNA could significantly inhibit the expression and function of MRP2 and P-gp, and the model of RNAi in vitro could be used to evaluate the role of efflux transporters in transportation of drugs.
