title: MiR-7-1 potentiated estrogen receptor agonists for functional neuroprotection in VSC4.1 motoneurons.
abstract: Protection of motoneurons is an important goal in the treatment of spinal cord injury (SCI). We tested whether neuroprotective microRNAs (miRs) like miR-206, miR-17, miR-21, miR-7-1, and miR-106a could enhance efficacy of estrogen receptor (ER) agonists such as 1,3,5-tris (4-hydroxyphenyl)-4-propyl-1H-pyrazole (PPT, ERα agonist), Way200070 (WAY, ERβ agonist), and estrogen (EST, ERα and ERβ agonist) in preventing apoptosis in the calcium ionophore (CI)-insulted ventral spinal cord 4.1 (VSC4.1) motoneurons. We determined that 200 nM CI induced 70% cell death. Treatment with 50 nM PPT, 100 nM WAY, and 150 nM EST induced overexpression of ERα, ERβ, and both receptors, respectively, at mRNA and protein levels. Treatment with ER agonists significantly upregulated miR-206, miR-17, and miR-7-1 in the CI-insulted VSC4.1 motoneurons. Transfection with miR-206, miR-17, or miR-7-1 mimic potentiated WAY or EST to inhibit apoptosis in the CI-insulted VSC4.1 motoneurons. Overexpression of miR-7-1 maximally increased efficacy of WAY and EST for down regulation of pro-apoptotic Bax and upregulation of anti-apoptotic Bcl-2. A search using microRNA database (miRDB) indicated that miR-7-1 could inhibit the expression of L-type Ca(2+) channel protein alpha 1C (CPα1C). miR-7-1 overexpression and WAY or EST treatment down regulated CPα1C but upregulated p-Akt to trigger cell survival signaling. The same therapeutic strategy increased expression of the Ca(2+)/calmodulin-dependent protein kinase II beta (CaMKIIβ) and the phosphorylated cAMP response element binding protein (p-CREB) so as to promote Bcl-2 transcription. Whole cell membrane potential and mitochondrial membrane potential studies indicated that miR-7-1 highly potentiated EST to preserve functionality in the CI-insulted VSC4.1 motoneurons. In conclusion, our data indicated that miR-7-1 most significantly potentiated efficacy of EST for functional neuroprotection and this therapeutic strategy could be used in the future to attenuate apoptosis of motoneurons in SCI.
authors.affiliation: Department of Pathology, Microbiology, and Immunology, University of South Carolina School of Medicine, Columbia, SC 29209, United States.
authors.forename: M
authors.initials: M
authors.lastname: Chakrabarti
authors.affiliation: Department of Neurosciences, Medical University of South Carolina, Charleston, SC 29425, United States.
authors.forename: N L
authors.initials: NL
authors.lastname: Banik
authors.affiliation: Department of Pathology, Microbiology, and Immunology, University of South Carolina School of Medicine, Columbia, SC 29209, United States. Electronic address: swapan.ray@uscmed.sc.edu.
authors.forename: S K
authors.initials: SK
authors.lastname: Ray
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002460:Cell Line; D002712:Chlorides / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D003576:Electron Transport Complex IV / Q000378:metabolism; D004622:Embryo, Mammalian; D004967:Estrogens / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D036145:Ginsenosides / Q000494:pharmacology; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D008564:Membrane Potentials / Q000187:drug effects; D035683:MicroRNAs / Q000494:pharmacology*; D009046:Motor Neurons / Q000187:drug effects*; D058632:Neuroprostanes / Q000494:pharmacology*; D010080:Oxazoles / Q000494:pharmacology; D010636:Phenols / Q000494:pharmacology; D051381:Rats; D011960:Receptors, Estrogen / Q000819:agonists* / Q000235:genetics / Q000378:metabolism; D012502:Sapogenins / Q000494:pharmacology; D013116:Spinal Cord / Q000166:cytology*
chemical_list: D002712:Chlorides; D004967:Estrogens; D036145:Ginsenosides; D035683:MicroRNAs; D058632:Neuroprostanes; D010080:Oxazoles; D010636:Phenols; D011960:Receptors, Estrogen; D012502:Sapogenins; C502077:WAY 200070; C507965:ginsenoside 20S-protopanaxatriol; D003576:Electron Transport Complex IV
keywords: 1,3,5-tris (4-hydroxyphenyl)-4-propyl-1H-pyrazole; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; CI; COX-4; CPα1C; CREB; Ca(2+)/calmodulin-dependent protein kinase II beta; CaMKIIβ; DMEM; DMSO; Dulbecco’s Modified Eagle Medium; ECL; ER agonists; ERα; ERβ; EST; FBS; FITC; GAPDH; HEPES; IgG; Immunoglobulin G; L-type Ca(2+) channel protein alpha 1C; MTT; PCR; PI; PPT; RT-PCR; SBDP; SCI; VSC4.1; VSC4.1 motoneurons; WAY; Way200070; apoptosis; cAMP response element binding protein; calcium ionophore; cytochrome c oxidase subunit IV; dimethyl sulfoxide; enhanced chemiluminescence; estrogen; estrogen receptor alpha; estrogen receptor beta; fetal bovine serum; fluorescein isothiocyanate; glyceraldehyde-3-phosphate dehydrogenase; hydroxyethyl piperazineethanesulfonic acid; miR-7-1; miRDB; miRs; microRNA database; microRNAs; p-CREB; phosphorylated cAMP response element binding protein; polymerase chain reaction; propidiun iodide; qRT-PCR; quantitative reverse transcription-polymerase chain reaction; reverse transcription-polymerase chain reaction; spectrin breakdown product; spinal cord injury; ventral spinal cord 4.1