Abstract
The aim of the present study was to specifically silence the rat ATP-binding cassette transporter G2 (rABCG2) gene in brain capillary endothelial cells by transfection of short interfering RNA (siRNA). Four different siRNAs designed to target rABCG2 were each transfected into HEK293 cells with myc-tagged rABCG2 cDNA. Quantitative real-time PCR and western blot analyses revealed that three of the siRNAs were able to reduce exogenous rABCG2 mRNA and protein levels in HEK293 cells. Moreover, rABCG2-mediated mitoxantrone efflux transport was suppressed by the introduction of these three siRNAs into HEK293 cells. In contrast, the other siRNA and non-specific control siRNA did not significantly affect the mRNA expression, the protein level or the transport activity. Endogenous rABCG2 mRNA and protein expression in a conditionally immortalized rat brain capillary endothelial cell line (TR-BBB13) was suppressed by the most potent siRNA among the four siRNAs tested. Furthermore, this siRNA did not affect the mRNA levels of other ABC transporters, such as ABCB1, ABCC1 and ABCG1, and the protein level of ABCB1 in TR-BBB13 cells, suggesting that it can selectively silence rABCG2 at the blood-brain barrier. This should be a useful and novel strategy for clarifying the contribution of rABCG2 to brain-to-blood transport of substrate drugs and endogenous compounds across the blood-brain barrier.
| Original language | English |
|---|---|
| Pages (from-to) | 63-71 |
| Number of pages | 9 |
| Journal | Journal of Neurochemistry |
| Volume | 93 |
| Issue number | 1 |
| DOIs | |
| Publication status | Published - 2005 Apr 1 |
| Externally published | Yes |
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Keywords
- 17β-estradiol
- ABC transporter
- ATP-binding cassette transporter G2
- Blood-brain barrier
- In vitro blood-brain barrier model
- Short interfering RNA
ASJC Scopus subject areas
- Biochemistry
- Cellular and Molecular Neuroscience
Cite this
Selective gene silencing of rat ATP-binding cassette G2 transporter in an in vitro blood-brain barrier model by short interfering RNA. / Hori, Satoko; Ohtsuki, Sumio; Ichinowatari, Masashi; Yokota, Takanori; Kanda, Takashi; Terasaki, Tetsuya.
In: Journal of Neurochemistry, Vol. 93, No. 1, 01.04.2005, p. 63-71.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Selective gene silencing of rat ATP-binding cassette G2 transporter in an in vitro blood-brain barrier model by short interfering RNA
AU - Hori, Satoko
AU - Ohtsuki, Sumio
AU - Ichinowatari, Masashi
AU - Yokota, Takanori
AU - Kanda, Takashi
AU - Terasaki, Tetsuya
PY - 2005/4/1
Y1 - 2005/4/1
N2 - The aim of the present study was to specifically silence the rat ATP-binding cassette transporter G2 (rABCG2) gene in brain capillary endothelial cells by transfection of short interfering RNA (siRNA). Four different siRNAs designed to target rABCG2 were each transfected into HEK293 cells with myc-tagged rABCG2 cDNA. Quantitative real-time PCR and western blot analyses revealed that three of the siRNAs were able to reduce exogenous rABCG2 mRNA and protein levels in HEK293 cells. Moreover, rABCG2-mediated mitoxantrone efflux transport was suppressed by the introduction of these three siRNAs into HEK293 cells. In contrast, the other siRNA and non-specific control siRNA did not significantly affect the mRNA expression, the protein level or the transport activity. Endogenous rABCG2 mRNA and protein expression in a conditionally immortalized rat brain capillary endothelial cell line (TR-BBB13) was suppressed by the most potent siRNA among the four siRNAs tested. Furthermore, this siRNA did not affect the mRNA levels of other ABC transporters, such as ABCB1, ABCC1 and ABCG1, and the protein level of ABCB1 in TR-BBB13 cells, suggesting that it can selectively silence rABCG2 at the blood-brain barrier. This should be a useful and novel strategy for clarifying the contribution of rABCG2 to brain-to-blood transport of substrate drugs and endogenous compounds across the blood-brain barrier.
AB - The aim of the present study was to specifically silence the rat ATP-binding cassette transporter G2 (rABCG2) gene in brain capillary endothelial cells by transfection of short interfering RNA (siRNA). Four different siRNAs designed to target rABCG2 were each transfected into HEK293 cells with myc-tagged rABCG2 cDNA. Quantitative real-time PCR and western blot analyses revealed that three of the siRNAs were able to reduce exogenous rABCG2 mRNA and protein levels in HEK293 cells. Moreover, rABCG2-mediated mitoxantrone efflux transport was suppressed by the introduction of these three siRNAs into HEK293 cells. In contrast, the other siRNA and non-specific control siRNA did not significantly affect the mRNA expression, the protein level or the transport activity. Endogenous rABCG2 mRNA and protein expression in a conditionally immortalized rat brain capillary endothelial cell line (TR-BBB13) was suppressed by the most potent siRNA among the four siRNAs tested. Furthermore, this siRNA did not affect the mRNA levels of other ABC transporters, such as ABCB1, ABCC1 and ABCG1, and the protein level of ABCB1 in TR-BBB13 cells, suggesting that it can selectively silence rABCG2 at the blood-brain barrier. This should be a useful and novel strategy for clarifying the contribution of rABCG2 to brain-to-blood transport of substrate drugs and endogenous compounds across the blood-brain barrier.
KW - 17β-estradiol
KW - ABC transporter
KW - ATP-binding cassette transporter G2
KW - Blood-brain barrier
KW - In vitro blood-brain barrier model
KW - Short interfering RNA
UR - http://www.scopus.com/inward/record.url?scp=16244415183&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=16244415183&partnerID=8YFLogxK
U2 - 10.1111/j.1471-4159.2004.02994.x
DO - 10.1111/j.1471-4159.2004.02994.x
M3 - Article
C2 - 15773906
AN - SCOPUS:16244415183
VL - 93
SP - 63
EP - 71
JO - Journal of Neurochemistry
JF - Journal of Neurochemistry
SN - 0022-3042
IS - 1
ER -