Identification Of Adenohypophysis-prefer MiR-7and MiR-375Target Genes In Cattle

MicroRNAs (miRNAs) are a class of endogenous~22nt, single-strandednon-coding RNAs that can play important regulatory roles in animals and plants bytargeting mRNAs for cleavage or translational repression. miRNAs play an importantrole in tne regulation of biology process, including cell proliferation、differentiation、apoptosis, neurogenesis, organism development and tumor metastasis and so on.Adenohypophysis is the ‘‘master’’ endocrine organ, thus far, only a few studies haveaddressed the involvement of miRNAs in the functions of the pituitary.The current research aimed to study the relative expression levels of miR-375、miR-7、miR-362-5p、miR-182、miR-137、miR-125a、miR-148a、miR-379、miR-30d、miR-143、miR-423-5p、miR-26a in the different tissues of cattle, in order to find theadenohypophysis-prefer or exclusively expressed miRNAs，and the target gene werepredicted by TargetScan、RNA22and other biology softwares. Then we identified thetarget genes which involved in pituitary function or reproductive traits byDual-Luciferase.The results indicate that miR-375、miR-7、miR-182、miR-362-5p、miR-137、miR-148a、miR-379、miR-30d、miR-143、423-5p、miR-26a all have different degreeof expression in the tissues. The relative expression levels of miR-375、miR-7、miR-182、miR-125a have high expression level in the adenohypophysis relatively,what is interesting is that the expression of miR-7and miR-375areadenohypophysis-prefer, compare to other tissues, the relative expression level hasextremely significant difference in adenohypophysis. So we chose miR-7andmiR-375to study the functions in the adenohypophysis.We find the binding sites for miR-7are located in mRNAs of BMPR2、PTGFRN、GnRHR、FSHβ、SSTR1、IL1β through bioinformatic analysis, similarly, the mRNAs of BMPR2and Leptin have the binding sites for miR-375,suggestingthese genes may be the target genes of miR-7or miR-375. Moreover, luciferasereporter assays show that miR-7specifically binds to the mRNA of PTGFRN andmiR-375binds to the mRNA of BMPR2to inhibit the expression of luciferase activity.Mutational plasmid and miRNA mimics co-transfection into NIH3T3cells, luciferasereporter assays show that the inhibition of luciferase activity are all greatly decreasedwhen introducing a3-5nt mismatch mutation into the seed region of mRNA, theinhibition of miR-7to luciferase activity from75%of the wild-type to26%andmiR-375has lost the inhibition for luciferase activity, suggesting that GTCTTCC isthe interaction sites of miR-7and PTGFRN target sequence, GAACAAA is theinteraction sites of miR-375and BMPR2target sequence.In conclusion, the expression patterns of miR-7and miR-375and other10kindsof miRNAs in different tissues of cattle have been successfully detected. The resultsindicate that the expression of miR-7and miR-375are adenohypophysis-prefer, andPTGFRN is identified as a target gene of miR-7and BMPR2is identified as a targetgene of miR-375in vitro. This research provides the basis for the further studies ofthe regulatory roles of miR-7and miR-375in pituitary development, hormonesecretion and other functions.