Differential Expression Of Some MiRNAs In The Pineal Gland Of Ovis Aries In Different Reproductive Status And Identification Of Target Interaction Between MiRNAs And AANAT

The microRNAs (miRNAs) are 21-to 24-nucleotide-long non-coding RNAs and silence gene expression post-transcriptionally by binding to the 3’-untranslated regions (UTRs) of target mRNAs, participating in physiological, biochemical and pathological processes. The pineal gland is an important part of the main physiological way in animal seasonal oestrus through the secretion of melatonin, and regulates hypothalamus gonadotropin-releasing hormone which shows pulse type release, further through the hypothalamus-pituitary-gonad axis control seasonal breeding activities. Among them, arylalkylamine-N-acetyltransferase (AANAT) is a key enzyme for the synthesis of melatonin, participates in regulating seasonal breeding for animals. This study aims to explore the differential expression of some miRNAs in the sheep pineal gland in different reproductive status and the epigenetic regulation function of miRNA to AANAT. According to the early result of sheep transcriptome sequencing, a part of miRNAs in the sheep pineal gland and AANAT expression in different reproductive status are identificated. Bioinformatics method is used to predict the candidate target relation between miRNAs and AANAT. Finally dual-luciferase system is usd to test and verify. The main results are as follows:1. Based on the RNA-seq result in the preliminary stage, real-time PCR is used to analysis the different expression of AANAT gene and five candidate miRNAs (miR-118,-247,-364,-406,-892) in the sheep pineal gland. The result indicates that AANAT gene expression level in anestrus is higher than in diestrus, up-regulate from diestrus to proestrus and reach the maximum in estrus. The changes of miR-364,-406,-892 expression has a contrary trend with the changes of AANAT gene.The expression quantity of miR-118 and miR-247 increases from anestrus to diestrus. decrease from diestrus to proestrus, and increase from proestrus to estrus. After analysis, it turns out that the expression level of AANAT and miR-364,-406 has negative correlation significantly (R2=0.572; P<0.05), and miR-892 has strong negative correlation significantly (R2=0.669; P< 0.05).2.3’RACE method is used to amplify the AANAT 3’-UTR and sequencing. Then miRanda and RNAhybrid bioinformatics software are used to predict candidate miRNAs which target AANAT 3’-UTR region, and the target combined position between miR-118,-247 and AANAT has been determined.3. Identification of target interaction between miR-118,-247 and AANAT. Synthetic wild type and mutant type sequence of AANAT 3’-UTR are built into pmirGLO vector, and synthetic the mature sequence of miR-118,-247 and negative control are built into pcDNA6.2 TM-GW/EmGFP-miR vector. The experiment were divided into 4 experimental groups (WT+miRNA, MUT+miRNA, WT+NE, MUT+NE) and a blank control group, which sets up three repeat in each group, and are transfected into 293 FT cell. The dual-luciferase report system shows that miR-118 inhibits luciferase activity of wild type AANAT 3’-UTR recombinant plasmid highly significantly (P<0.01), means interaction between miR-118 and AANAT 3’-UTR and confirms target site of interaction, proves the existence target relationship between miR-118 and AANAT. However, miR-247 does not inhibit AANAT significantly, suggests that target relationship between miR-247 and AANAT does not exist.