microRNA expression during ovine adult mammary gland development

The mammary gland undergoes extensive remodeling between the beginning of pregnancy and lactation; this involves cellular processes including cell proliferation, differentiation, and apoptosis, all of which are under the control of numerous regulators. To unravel the role played by miRNA, we describe here 47 new ovine miRNA cloned from mammary gland in early pregnancy displaying strong similarities with those already identified in the cow, human, or mouse. A microarray study of miRNA variations in the adult ovine mammary gland during pregnancy and lactation showed that 100 miRNA are regulated according to three principal patterns of expression: a de- crease in early pregnancy, a peak at midpregnancy, or an increase throughout late pregnancy and lactation. One miRNA displaying each pattern (miR-21, miR-205, and miR-200b) was analyzed by qRT- PCR. Variations in expression were confirmed for all three miRNA. Using in situ hybridization, we detected both miR-21 and miR-200 in luminal mammary epithelial cells when expressed, whereas miR-205 was expressed in basal cells during the first half of pregnancy and then in luminal cells during the second half. We therefore conclude that miR-21 is strongly expressed in the luminal cells of the normal mammary gland during early pregnancy when extensive cell proliferation occurs. In addition, we show that miR-205 and miR-200 are coexpressed in luminal cells, but only during the second half of pregnancy. These two miRNA may cooperate to maintain epithelial status by repressing an EMT-like program, to achieve and preserve the secretory phenotype of mammary epithelial cells. 5 samples for sheep and 5 samples for mouse乳腺在妊娠起始至泌乳阶段会经历广泛的重塑过程，这一过程涉及细胞增殖、分化与凋亡等细胞生物学事件，所有这些过程均受众多调控因子的精密调控。为阐明微小RNA（miRNA）在此过程中的作用，本文报道了从妊娠早期绵羊乳腺中克隆得到的47种新型绵羊微小RNA，这些miRNA与已在牛、人或小鼠中鉴定出的同类miRNA具有高度同源性。针对成年绵羊乳腺在妊娠与泌乳阶段的微小RNA表达变化开展的基因芯片研究显示，共有100种微小RNA的表达可归纳为三种主要模式：妊娠早期表达下调、妊娠中期表达达到峰值，或在妊娠晚期及泌乳阶段持续上调。针对每种表达模式各选取一种微小RNA（miR-21、miR-205与miR-200b），通过实时定量荧光PCR（qRT-PCR）进行验证，结果确认了这三种微小RNA的表达变化趋势。利用原位杂交（in situ hybridization）技术，我们检测到当miR-21与miR-200表达时，它们定位于乳腺腔上皮细胞；而miR-205则在妊娠前半段表达于基底细胞，在妊娠后半段则转移至腔上皮细胞中表达。据此我们得出结论：在细胞大量增殖的妊娠早期，miR-21在正常乳腺的腔上皮细胞中呈高表达。此外，我们发现miR-205与miR-200仅在妊娠后半段的腔上皮细胞中共表达，这两种微小RNA可能通过抑制上皮间质转化（EMT）样程序来维持上皮细胞状态，从而实现并维持乳腺上皮细胞的分泌表型。本研究包含5份绵羊样本与5份小鼠样本。