MiR-138 indirectly regulates the MDR1 promoter by NF-κB/p65 silencing.
MicroRNAs (miRNAs) are known to mediate post-transcriptional gene silencing in the cytoplasm and recent evidence indicates that may also possess nuclear roles in regulating gene expression. A previous study showed that miR-138 is involved in the multidrug resistance of leukemia cells through down-regulation of the drug efflux pump P-glycoprotein (P-gp), the protein encoded by the human multidrug-resistant ABCB1/MDR1 gene. However, the transcriptional regulatory mechanisms responsible remain to be elucidated. To deepen the description of the mechanism of transcriptional gene silencing on the MDR1 promoter, we initially performed a bioinformatics search for potential miR-138 binding sites in the MDR1 gene promoter sequence. Interestingly, we did not find miR-138 binding sites in this region, suggesting an indirect regulation. From six representative transcriptional factors involved in MDR1 gene regulation, an in silico analysis revealed that NF-κB/p65 has a specific binding site for miR-138. The results of luciferase reporter assay, western blot and flow cytometry shown here suggest that miR-138 might modulate the human MDR1 expression by inhibiting NF-κB/p65 as an indirect mechanism of MDR1 regulation. Furthermore, employing the human macrophage-like cell line U937 we observed comparable results with NF-κB/p65 down-regulation and we also observed a significant reduction in the IL-6 and TNF-α mRNA, as well as in their secreted pro-inflammatory cytokines following miR-138 expression, suggesting that canonical NF-κB target genes might also be potential targets for miR-138 in leukemia cells.
Laboratorio de Investigación en Genómica, Genética y Bioinformática, Hospital Infantil de México, Federico Gómez, Mexico City, Mexico; Posgrado en Ciencias Biológicas, Universidad Nacional Autónoma de México, Mexico.
J L
JL
Requenez-Contreras
Laboratorio de Investigación en Genómica, Genética y Bioinformática, Hospital Infantil de México, Federico Gómez, Mexico City, Mexico; Posgrado en Ciencias Biológicas, Universidad Nacional Autónoma de México, Mexico.
E S
ES
López-Castillejos
Laboratorio de Investigación en Genómica, Genética y Bioinformática, Hospital Infantil de México, Federico Gómez, Mexico City, Mexico; Posgrado en Ciencias Biológicas, Universidad Nacional Autónoma de México, Mexico.
R
R
Hernández-Flores
Laboratorio de Inmunobiología, Hospital Juárez de México, Mexico.
M A
MA
Moreno-Eutimio
Laboratorio de Investigación en Genómica, Genética y Bioinformática, Hospital Infantil de México, Federico Gómez, Mexico City, Mexico.
J T
JT
Granados-Riveron
Unidad de Investigación en Enfermedades Oncológicas, Hospital Infantil de México, Federico Gómez, Mexico.
G U
GU
Martinez-Ruiz
Laboratorio de Investigación en Genómica, Genética y Bioinformática, Hospital Infantil de México, Federico Gómez, Mexico City, Mexico. Electronic address: guillaqui@himfg.edu.mx.
G
G
Aquino-Jarquin
D018435:ATP Binding Cassette Transporter, Subfamily B / Q000235:genetics; D002352:Carrier Proteins / Q000235:genetics*; D002478:Cells, Cultured; D003198:Computer Simulation; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D035683:MicroRNAs / Q000235:genetics*; D008957:Models, Genetic; D016328:NF-kappa B / Q000235:genetics*; D009363:Neoplasm Proteins / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D034622:RNA Interference / Q000502:physiology*
C513055:ABCB1 protein, human; D018435:ATP Binding Cassette Transporter, Subfamily B; D002352:Carrier Proteins; C484020:HSAJ2425 protein, human; D047908:Intracellular Signaling Peptides and Proteins; C546110:MIRN138 microRNA, human; D035683:MicroRNAs; D016328:NF-kappa B; D009363:Neoplasm Proteins
Indirect mechanism; MDR1 promoter; NF-κB/p65 protein; RelA mRNA; miR-138