Overexpression of miR-99a in hippocampus leads to impairment of reversal learning in mice.

As one of the most common human genetic disorders, Down syndrome (DS) is characterized by a mild-to-moderate cognitive disability, which mainly results from genes overexpression on chromosome 21. The expression of miR-99a, a gene harboring on chromosome 21, is increased by 50 folds in DS brain samples. This study aims to investigate the effect of miR-99a overexpression in the hippocampus on mouse behaviors and explore the underlying mechanisms. Lentivirus vectors were delivered into the hippocampus for focal miR-99a overexpression in mice. Then behaviors were observed by an open field, elevated plus maze, rotarod motor test, and Morris water maze. The genes affected by miR-99a were identified by RNA sequencing (RNA-seq) and confirmed by quantitative RT-PCR (qRT-PCR) in samples isolated from the hippocampus injected with lentivirus-GFP-miR-99a or lentivirus-GFP vectors. It was found that the expression of miR-99a with intrahippocampal delivery of lentivirus-GFP-miR-99a resulted in reversal learning impairment in mice although it had no influence on motor function and anxiety. Meanwhile, RNA-seq results showed that 92 genes including mRNAs and microRNAs were significantly regulated by miR-99a, consistent with qRT-PCR consequence. Moreover, dual-luciferase reporter assay showed that miR-99a could directly bind to the 3'-untranslated regions (3'UTR) of target genes (Clic6 and Kcnj13) with an inhibitory effect on their activity. Furthermore, we also found that miR-99a overexpression affected different biological processes by bioinformatic analyses. Our study showed that miR-99a overexpression in the hippocampus leads to cognitive impairment through regulating the expressions of various genes, which reveals a novel function of miR-99a and provides new insights into understanding the pathophysiologic process of DS.