microRNA-145-5p regulates P-glycoprotein and breast cancer resistance protein in primary human fetal brain endothelial cells.

P-glycoprotein (P-gp, encoded by ABCB1) and breast cancer resistance protein (BCRP/ABCG2), present in the blood-brain barrier, protect the fetal brain from exposure to neurotoxicants. P-gp and BCRP are modulated by microRNA-145-5p (miR-145-5p) in cancer cells. In the present study, we hypothesized that miR-145-5p modulates P-gp and BCRP in human fetal brain endothelial cells (hfBECs) and in an adult BEC line (human cerebral microvascular endothelial cell line; hCMEC/D3). miR-145-5p mimic and inhibitor were transfected into hfBECs (n = 6, mid-gestation) and hCMEC/D3 cells. P-gp and BCRP mRNA, protein expression and efflux activity were assessed using qPCR, Western blotting, immunofluorescence and calcein-AM and chlorin e6 assays. Target specificity was evaluated using luciferase reporter assays. hfBECs exhibited lower ABCB1 and ABCG2 mRNA and protein expression and reduced P-gp activity, but significantly increased BCRP activity, compared with hCMEC/D3 cells. The miR-145-5p mimic reduced luciferase activity of an ABCB1 3'-untranslated region (3'-UTR) reporter but had no effect on the ABCG2 3'-UTR. In hfBECs, miR-145-5p overexpression reduced P-gp and BCRP levels and efflux activity. In contrast, miR-145-5p overexpression in hCMEC/D3 cells increased P-gp activity while reducing BCRP activity. Inhibition of miR-145-5p in hfBECs increased P-gp protein levels and exhibited a trend towards increased P-gp activity, without altering BCRP levels, although BCRP activity significantly increased. miR-145-5p directly modulates the expression and activity of P-gp and indirectly modulates BCRP in fetal and adult human BECs. These data suggest that miR-145-5p plays an important role in the regulation of key drug transporters at the BBB in fetal brain and may modulate BBB function in adulthood. KEY POINTS: microRNA-145-5p (miR-145-5p) directly targets the 3'-untranslated region (3'-UTR) of ABCB1, but not ABCG2, as shown by luciferase assays. In human fetal brain endothelial cells (hfBECs), miR-145-5p mimic reduces P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) levels and transporter activity. Inhibition of miR-145-5p in hfBECs increases P-gp and BCRP activities. In adult human cerebral microvascular endothelial cell line (hCMEC/D3) cells, miR-145-5p mimic increases P-gp activity and decreases BCRP activity, indicating developmental differences in regulation. miR-145-5p directly modulates P-gp and indirectly modulates BCRP, suggesting a key role in blood-brain barrier (BBB) transporter regulation during fetal development and potentially in adulthood.