Differentially MicroRNAs Expression Analysis In Sexual Mature And Immature Bovine Pituitary And Their Functional Study

MicroRNA (miRNA) is a novel class of endogenous non-codingposttranscriptional regulatory small RNA, which could inhibit mRNA translation orcause mRNA degradation by interacting with their target genes in the3′-UTR ofmRNA. Studies have shown that miRNA regulates more than30%of theprotein-coding genes, thereby identification and functional analysis of miRNAs havebecome a hot topic in molecular biology. Pituitary is an important endocrine organ inanimals, and it secretes a variety of hormones and plays an important role in theregulation of animal life, of which pituitary gonadotropins luteinizing hormone andfollicle-stimulating hormone play a major role on reproduction. They functionsynergistically, which is instrumental in adjusting the development of spermatogeniccells and mesenchymal cells, synthesizing and secreting the gonadal steroid hormones,maintaining normal spermatogenesis function in males. It is possible that miRNAaffects the synthesis and secretion by regulating FSH, LH gene transcription andtranslation, thus affecting the ability of testicular endocrine function andspermatogenesis of males.Cattle is an important herbivorous livestock，and molecular breeding research isone of the hot topics in recent years. However, the current research on bovine miRNAis far behind the rats, mice and other species. The main reason is that there are stillsome limitations by applying the traditional experimental methods and bioinformaticsmethods in the identification of cattle miRNA. The next-generation sequencing (NGS)provides a new sequencing platform for bovine miRNA research. However, there arestill not many research reports on bovine reproductive traits related miRNA.This research is performed on pituitary tissue of before and after Sexualmaturation in Yanbian cattle. Through the construction of cDNA library of pituitarytissue miRNA, using the Solexa high-throughput sequencing technology combinedwith bioinformatics analysis to identify miRNA expression profiling of bovinepituitary tissue, variance analysis, the use of bioinformatics software to predict miRNA target genes differential expression and functional analysis, further selectingsome differential expression miRNA, GO and KEGG pathway analysis, verifying therelevant miRNA target genes, the establishment of miRNA differential expressionprofiling of rat anterior pituitary cells in different developmental stages, and theanalysis of conservative bovine pituitary miRNA, improving rat pituitary cell isolationand culture system, verifying miRNA target gene regulatory functions of primary cells,the results can be summarized as followings:1. Using the high-throughput sequencing technology, Solexa,we succeed inestablishing the differential expression profiles of1month,24-month-old Yanbian inbovine pituitary tissue and identified347mature bovine miRNAs as well as187newcandidate bovine miRNAs.There was279known or conserved miRNAs expresseddifferentially in the miRNA expression profiles of1month and24-month-old bovinepituitaries.While90were significantly up-regulated among them,129weredown-regulated. The fluorescent quantitative validation results and Solexa sequencinganalysis resulted in the same change trend.2.12942different target genes were predicted to be targeted by differentiallyexpressed miRNAs predicted. GO analysis showed that were collected by34clustering related propagation and hormone secretion, and relevant target genes were74KEGG analysis identified71Pathway.3. We established the miRNA differentially expression profiles of21-day-old and6-month-old rat pituitary. The chip detected93known and conserved miRNAsdifferentially expressed, of which57down-regulated,36up-regulated. And we found51miRNAs were conserved both in the rat and bovine pituitary,21miRNAs amongthem and the bovine miRNA expression profile resulted in the same change trend.4. We predicted miR-23a target gene FSHR, and successfully constructed themutant plasmids and recombinant plasmids that containing miR-23a complementarybinding sequence pmirGLO-FSHR, and the sequence complementary to a miRNAseed AATGTGA was mutated to AATCTCA, dual luciferase reporter systemverifiedthat miR-23can inhibit the expression of FSHR, the seed sequence of miR-23a wasUCACAUU.5. Optimize the isolation and culture system of rat anterior pituitary cells.Cellscultured for4days replaced serum-free medium,the cultured to7days cellsgrowedin good condition.Rat pituitary cells transfected with miR-23a mimics after48 hours, FSH secretion was not affected (P>0.05). The expression of FSHR genedecreased significantly (P <0.01), proving that FSHR was the target gene of miR-23a.