title: shRNA-induced saturation of the microRNA pathway in the rat brain.
abstract: RNA interference (RNAi) is a powerful strategy for unraveling gene function and for drug target validation, but exogenous expression of short hairpin RNAs (shRNAs) has been associated with severe side effects. These may be caused by saturation of the microRNA pathway. This study shows degenerative changes in cell morphology and intrusion of blood vessels after transduction of the ventromedial hypothalamus (VMH) of rats with a shRNA expressing adeno-associated viral (AAV) vector. To investigate whether saturation of the microRNA pathway has a role in the observed side effects, expression of neuronal microRNA miR-124 was used as a marker. Neurons transduced with the AAV vector carrying the shRNA displayed a decrease in miR-124 expression. The decreased expression was unrelated to shRNA sequence or target and observed as early as 1 week after injection. In conclusion, this study shows that the tissue response after AAV-directed expression of a shRNA to the VMH is likely to be caused by shRNA-induced saturation of the microRNA pathway. We recommend controlling for miR-124 expression when using RNAi as a tool for studying (loss of) gene function in the brain as phenotypic effects caused by saturation of the RNAi pathway might mask true effects of specific downregulation of the shRNA target.
authors.affiliation: Department of Neuroscience and Pharmacology, Brain Center Rudolf Magnus, University Medical Center Utrecht, Utrecht, The Netherlands.
authors.forename: M A
authors.initials: MA
authors.lastname: van Gestel
authors.affiliation: Department of Neuroscience and Pharmacology, Brain Center Rudolf Magnus, University Medical Center Utrecht, Utrecht, The Netherlands.
authors.forename: S
authors.initials: S
authors.lastname: van Erp
authors.affiliation: Department of Neuroscience and Pharmacology, Brain Center Rudolf Magnus, University Medical Center Utrecht, Utrecht, The Netherlands.
authors.forename: L E
authors.initials: LE
authors.lastname: Sanders
authors.affiliation: Department of Neuroscience and Pharmacology, Brain Center Rudolf Magnus, University Medical Center Utrecht, Utrecht, The Netherlands.
authors.forename: M A D
authors.initials: MA
authors.lastname: Brans
authors.affiliation: Department of Neuroscience and Pharmacology, Brain Center Rudolf Magnus, University Medical Center Utrecht, Utrecht, The Netherlands.
authors.forename: M C M
authors.initials: MC
authors.lastname: Luijendijk
authors.affiliation: Department of Neuroscience and Pharmacology, Brain Center Rudolf Magnus, University Medical Center Utrecht, Utrecht, The Netherlands.
authors.forename: M
authors.initials: M
authors.lastname: Merkestein
authors.affiliation: Department of Neuroscience and Pharmacology, Brain Center Rudolf Magnus, University Medical Center Utrecht, Utrecht, The Netherlands.
authors.forename: R J
authors.initials: RJ
authors.lastname: Pasterkamp
authors.affiliation: Department of Neuroscience and Pharmacology, Brain Center Rudolf Magnus, University Medical Center Utrecht, Utrecht, The Netherlands.
authors.forename: R A H
authors.initials: RA
authors.lastname: Adan
mesh_terms: D000818:Animals; D000229:Dependovirus / Q000235:genetics; D005786:Gene Expression Regulation; D005822:Genetic Vectors / Q000008:administration & dosage / Q000009:adverse effects*; D035683:MicroRNAs / Q000235:genetics* / Q000633:toxicity; D009474:Neurons / Q000378:metabolism*; D009928:Organ Specificity; D034741:RNA, Small Interfering / Q000235:genetics / Q000494:pharmacology*; D051381:Rats; D017208:Rats, Wistar; D015398:Signal Transduction / Q000187:drug effects*; D014697:Ventromedial Hypothalamic Nucleus / Q000378:metabolism
chemical_list: C548144:MIRN124 microRNA, rat; D035683:MicroRNAs; D034741:RNA, Small Interfering