How to Explore the Function and Importance of MicroRNAs: MicroRNAs Expression Profile and Their Target/Pathway Prediction in Bovine Ovarian Cells
Micro RNAs (miRNA) are integral components of genetic regulatory networks and act by binding to the transcripts of their corresponding target genes, leading to a decrease in protein production levels either by mRNA degradation or by translational repression. While the role of miRNAs is ubiquitous, they have a particular importance with regard to cell differentiation. The miRNA-target mRNA interaction has a significant impact on many signaling pathways and the cross-talk between them; playing a regulatory role in a variety of different physiological processes within the cells. Ovarian follicle development is a physiological process that is not fully understood with regard to miRNA regulation; there are many questions that remain with respect to the molecular regulation of this important process. Bovine follicular cells are a good experimental model for the investigation of these mechanisms, having direct implications on reproductive health in humans. This chapter describes how differentially expressed miRNAs are identified in the granulosa and theca cells of dominant and subordinate bovine ovarian follicles and the identification of their associated targets and pathways. This chapter systematically describes how the granulosa and theca cells are dissected from the ovarian follicles. Afterward, we present a detailed protocol for miRNA extraction, based on a combined TRI reagent/column clean-up method, and also miRNA expression profiling using both microarray and RT-qPCR. In addition, an outline is provided of the bioinformatic analysis which enables the prediction of miRNAs targets. Pathways associated with the differentially expressed miRNAs are also elucidated using DIANA-miRPath software.
Key wordsMicroRNAs expression Target/pathway prediction DIANA-miRPath Ovarian follicles Granulosa cells Theca cells Bovine
- 5.Mihm M, Baker PJ, Ireland JL, Smith GW, Coussens PM, Evans AC, Ireland JJ (2006) Molecular evidence that growth of dominant follicles involves a reduction in follicle-stimulating hormone dependence and an increase in luteinizing hormone dependence in cattle. Biol Reprod 74:1051–1059CrossRefPubMedGoogle Scholar
- 7.Forde N, Mihm M, Canty MJ, Zielak AE, Baker PJ, Park S, Lonergan P, Smith GW, Coussens PM, Ireland JJ, Evans ACO (2008) Differential expression of signal transduction factors in ovarian follicle development: a functional role for betaglycan and FIBP in granulosa cells in cattle. Physiol Genomics 33:193–204CrossRefPubMedGoogle Scholar
- 8.Walsh SW, Mehta JP, McGettigan PA, Browne JA, Forde N, Alibrahim RM, Mulligan FJ, Loftus B, Crowe MA, Matthews D, Diskin M, Mihm M, Evans AC (2012) Effect of the metabolic environment at key stages of follicle development in cattle: focus on steroid biosynthesis. Physiol Genomics 44:504–517CrossRefPubMedGoogle Scholar
- 18.Vlachos IS, Zagganas K, Paraskevopoulou MD, Georgakilas G, Karagkouni D, Vergoulis T, Dalamagas T, Hatzigeorgiou AG (2015) DIANA-miRPath v3.0: deciphering microRNA function with experimental support. Nucleic Acids Res 43:W460–W466. https://doi.org/10.1093/nar/gkv403 CrossRefPubMedPubMedCentralGoogle Scholar