Sex-Dependent Changes of miRNA Levels in the Hippocampus of Adrenalectomized Rats Following Acute Corticosterone Administration

We explored sex-biased effects of the primary stress glucocorticoid hormone corticosterone on the miRNA expression profile in the rat hippocampus. Adult adrenalectomized (ADX) female and male rats received a single corticosterone (10 mg/kg) or vehicle injection and after 6 h, hippocampi were collected for miRNA, mRNA and Western blot analyses. miRNA profiling microarrays showed a basal sex-biased miRNA profile in ADX rat hippocampi. Additionally, acute corticosterone administration triggered a sex-biased differential expression of miRNAs derived from genes located in several chromosomes and clusters on the X and 6 chromosomes. Putative promoter analysis unveiled that most corticosterone-responsive miRNA genes contained motifs for either direct or indirect glucocorticoid actions in both sexes. The evaluation of transcription factors indicated that almost 50 % of miRNA genes sensitive to corticosterone in both sexes was under glucocorticoid receptor regulation. Transcription factor-miRNA regulatory network analyses identified several transcription factors that regulate, activate or repress miRNA expression. Validated target mRNA analysis of corticosterone-responsive miRNAs showed a more complex miRNA-mRNA interaction network in males compared to females. Enrichment analysis revealed that several hippocampal-relevant pathways were affected in both sexes, such as neurogenesis and neurotrophin signaling. The evaluation of selected miRNA targets from these pathways displayed a strong sex difference in the hippocampus of ADX-vehicle rats. Corticosterone treatment did not change the levels of the miRNA targets and their corresponding tested proteins. Our data indicate that corticosterone exerts a sex-biased effect on hippocampal miRNA expression, which may engage in sculpting the basal sex differences observed at higher levels of hippocampal functioning. Male (n=9) and female (n=9) rats underwent bilateral adrenalectomy (ADX) surgery under isofluorane anesthesia. Seven days after surgery, six ADX animals from each sex were injected with CORT previously dissolved in ethanol and then diluted in saline (10 mg/kg, i.p.). The remaining ADX animals were injected with vehicle (ethanol solution diluted in saline). Considering that GCs promote genomic actions, we chose to euthanize the animals 6 h post-injection as a period in which it is highly probable to observe the effects of the hormone. At the time of sacrifice, trunk blood was collected for the determination of serum CORT levels. Hippocampi were rapidly dissected and flash-frozen in liquid nitrogen and kept at -80 °C until processing for RNA isolation. Frozen hippocampal tissue was disrupted using an UltraTurrax homogenizer in QIAzol Lysis Reagent (Qiagen, California, USA). Enriched RNA fractions of less than 200 nucleotides were then obtained by using the RNeasy Mini Kit (Qiagen, California, USA), followed by the RNeasy MinElute Cleanup Kit After labeling the RNAs with the Affymetrix FlashTagTMBiotin H.S.R. kit, samples were hybridized on Affymetrix GeneChipTM Version 3 miRNA arrays (680 mature and 486 pre-miRNA rat probe sets), according to the Affymetrix hybridization protocols. Array slides were then stained with streptavidin/phycoerythrin, and after washing, they were scanned using the Affymetrix GCS 3000 7G and GeneChip® Operating Software (AGCC; Version 3.2). We used 3 experimental replicas for each sex, including ADX-vehicle and CORT administration.