Identification And Biological Role Analysis Of MicroRNAs In Goat Ovaries

Goats (Capra hircus) are one of the important economical animals, as they providehigh-quality wool, meat and other products. However, hircine fecundity is relatively low,and this is a major constraint which prevents the development of the goat industry.Therefore, new methods to improve hircine fertility are highly desirable. Ovaries are oneof the important reproductive organs of animals, as they play a very important role inreproductive regulation.MicroRNAs (miRNAs) are a group of noncoding RNAs which are involved inovarian function and reproductive process by pairing to mRNAs which mainly results intarget-specific post-transcriptional repression. Although predictably important, themiRNAs of goat ovaries have been largely undefined and the indentification of them willbe helpful to facilitate studies on the regulation of miRNAs during mammalianreproduction and goat molecular breeding.There are significant differences in the activity and endocrine characteristics of theovary during pregnancy and non-pregnancy. In the present study, we characterized andinvestigated the differential expression of miRNAs in the ovaries of pregnant andnon-pregnant goats using deep sequencing technology. Analysis of tissue expressionpattern and target gene function for differential miRNAs were performed with the purposeof further understanding the role of miRNAs in reproductive biological processes, and alsomay help to identify miRNAs which could be potentially used to regulate hircinereproduction and breeding practice in the future. The main points are indicated as follows:(1) A total of9,977,224and11,209,532raw reads were identified in the pregnant andnon-pregnant goat ovarian libraries, respectively. After data cleaning, a total of9,231,324and11,010,206clean reads associated with377,973and204,342unique sequences wereobtained from the two libraries. The majority of the small RNAs were21~24nt in size.Sequences22nt in length, the typical size of Dicer-derived products, accounted for42.54%and56.13%of the total sequence reads in the pregnant and non-pregnant ovarianlibraries. The results showed that miRNAs were widely expressed in goat ovaries.(2) After aligning the sequence data to miRBase, sheep genome and goat ESTsequences, a total of535and508conserved miRNAs of Ovis aries, Bos taurus, Sus scrofa,Equus caballus and Canis familiaris were identified in the pregnant and non-pregnantlibraries, respectively. After grouping the identical sequences, a total of617unique miRNAs were obtained from both libraries and the85sheep known miRNAs containedtherein. MiRNA expression is more concentrated and total expression levels of the top20miRNAs account for more than80%of the total expression levels of all miRNAs in eachlibrary. MiR-143and let-7b are the most highly expressed miRNA in the ovaries ofpregnant and non-pregnant goats, respectively. Moreover,7putative goat miRNAs werepredicted in our study.Considering locations on sheep chromosome of the top10miRNAs in each library,some of them had only one genomic position and others had two genomic positions. Fourkinds of combination of two miRNAs were identified to putative miRNA clusters.Sequencing analysis indicated the details of goat ovarian miRNAs and the isomiRs andmirSNPs contained therein. These sequence identity of the isomiR and mirSNP has broadand significant biological implications and there is no doublt that certain isomiRs andmirSNPs are signatures of specific biogenesis processes and/or functions, which until nowhave been largely unidentified.(3) A total of471within617conserved miRNAs were co-expressed in both libraries,and90pregnancy-specific and56non-pregnancy-specific miRNAs were identified.Additionally,294miRNAs were upregulated and113miRNAs were downregulated in thepregnant library compared to the non-pregnant library. The ten most highly expressedmiRNAs in the non-pregnant library were all downregulated in the pregnant library, and ofthe ten most highly expressed miRNAs in the pregnant library, six miRNAs (miR-143,miR-99a, miR-125b, miR-148a, miR-10b and miR-26a) and four miRNAs (let-7a, let-7f,let-7c and let-7b) were down-and upregulated in the non-pregnant library, respectively.(4) Using q-PCR to detect expression stability of8candidate reference genes across10kinds of goat tissues, the results indicated that the18S is the optimal reference gene forrelative quantitative analysis of gene expression in goat tissues under normal conditions.However, if higher accuracy requirements of the experiments, the18S and TBP or18S,TBP and HMBS reference combinations should be used.(5) Q-PCR analysis data of5randomly selected miRNAs were consistent with thesequencing data in pregnant and non-pregnant goat ovaries. Moreover, the miR-143andlet-7b were both widely expressed in9specific goat tissues and high expressed in goatreproductive organs such as ovary, oviduct and uterus.(6) The GO and KEGG pathway analysis indicated that the target genes of top10miRNA in each library were enriched in signaling pathways with reproductive processesincluding the GnRH, Wnt, MAPK, TGF-beta and oocyte meiosis. Moreover, the target genes of miR-143and let-7b were enriched in biological processes of regulation of cellproliferation and apoptosis, nucleotide binding, response to steroid hormone stimulus,regulation of phosphorylation, post transcriptional regulation of gene expression,regulation of transferase activity and so on. The evidences based on expression profile andfunctional analysis demonstrated that the miR-143and let-7b at least participate inregulation of goat reproductive process.