Identification Of Differentially Expressed MiRNAs And Their Targets In The Pre-ovulatory Follicles Of Large White And Erhualian Sows

miRNAs are 18-22nt endogenous, conservative, non-coding RNA, which widely exist in eukaryote. They can inhibit the translation or cleave messenger RNA (mRNA) by binding 3’UTR of target mRNAs, thereby affecting a series of biological processes. Many studies show that miRNAs are of great importance in animal ovarian follicle development, hormone modulation, cell apoptosis, follicular atresia. However, the molecular mechanism of miRNAs leading to the different performance in reproduction between different breeds has not been fully understood. In addition, the reports remain limited in pig miRNA target genes related to reproduction traits of pigs. Therefore, differentially expressed miRNAs and their targets between Large White and Erhualian pig are worthy to be identified. Our research is constituted by identifying the differentially expressed miRNAs between Large White and Erhualian ovarian follicles of pigs by Solexa deep sequencing and Real-time PCR, targets discovering of the differentially expressed miRNAs by degradome sequencing and combined target prediction software, validating the target genes by dual-luciferase reporter system, over expression and inhibition of miR-144. The results are as follows:1. Identification and verification of differentially expressed miRNAs between Large White and Erhualian ovarian follicles of pigs by Solexa deep sequencing and Real-time PCRRNA from ovarian follicles of Large White and Erhualian pigs was submitted to Solexa deep sequencing.1349 mature miRNAs were present and 390 of them were differentially expressed between Large White and Erhualian sows.124 miRNAs were up-regulated in Erhualian pig and the others were down regulated. QPCR was employed to detect six differentially expressed miRNAs including let-7a, miR-125a, miR-3613-5p, miR-331*, miR-2423 and miR-4028-3p, and the results were consisted with the sequencing data.2. Degradome sequencing combined with target prediction software to discover the targets of differentially expressed miRNAs581 miRNA-mRNA were discoveried by degradome sequencing of Erhualian pigs and PairFinder&t-plot analysis. Targets included 70 unique genes.2 target genes were pseudogenes and 12 of them were derived from mitochondrial genome. Gene Ontology and pathway analysis of target genes by DAVID showed that they were mainly involved in electron transportation, Oxidative phosphorylation. Cytoscape was employed to construct the network of differentially expressed miRNAs and their targets.PTGS2, MAK and CFTR gene were predicted to be the targets of miR-144 which was 46-fold higher in Large White by TargetScan, PicTar and miRanda.3. Target genes verification by Dual-Luciferase Detection SystemThe target sequences of MT-CO3, MT-CYB, MT-ND3, PTGS2, MAK and CFTR gene were amplified by PCR. Then they were linked to the pmirGLO vector and co-transfected with miRNA mimic to the Chinese Hamster Ovary Cell. Then we detected the interaction between miRNAs and their targets by Dual-Luciferase Detection System. The results showed that miR-3115 and miR-331* could inhibit the luciferase activity of MT-CO3 and MT-CYB respectively, and miR-144 was a negative regulator of PTGS2 and CFTR gene.4. Over expression and inhibition of miR-144Pig Kidney Cell was transfected with miRNA mimic and inhibitor of miR-144. miRNA and mRNA were reverse transcribed by RT-loop and random premier respectively. And then qPCR was used for quantitation. The results showed that the mRNA level of PTGS2 and CFTR were significantly down regulated in over expression experiment of miR-144 and up-regulated in inhibition experiment. However, the mRNA level of MAK was not significantly changed in these experiments.