The In Vivo Function Of Progestogen Responsive MiR-31 In Mammary Gland Development

The mammary gland is responsible for secreting milk to nourish the newborn offspring.During embryogenesis, mammary gland generates a rudimentary ductal system. At puberty, the mammary gland undergoes a rapid growth, in response to hormone filling the fat pad with mammary ducts. During th pregnancy, the mammary gland undergoes tremendous changes, the second and tertiary ductal branches increased then the alveolar buds differentiate to form alveoli.By the late pregnancy, the alveoli encompass the fat pad, which are competent to secret milk.After lactation, the gland involutes, going back to a pre-pregnant state. The mammary glands are used as a good model system to study adult stem cells, as it primarily develops postnatal is convenient to manipulate in vivo and in vitro. MicroRNA (miRNA) are small, non-coding RNA(20-23 nt in length) that post-transcriptionally regulate gene expression in plants and animals.Many in vitro studies have implicated that miRNAs are involved in the mammary gland development and mammary stem cells (MaSCs) self-renewal. However, the in vivo functions of specific miRNA in controlling mammary development and the behavior of MaSCs are poorly understood.In this study, we found that the expression level of miR-31 change with the stage of mammary gland development, gradually increases from puberty, and peaks at pregnancy stage.The dynamic expression pattern raised the possibility that miR-31 is regulated by hormones. We identified potential binding sites of transcription factors in the promoter using JASPAR database.Interestingly the promoter of miR-31 contains potential binding site of ERa and NF-?B (p65). The chromatin immunopreciptation assay showed that ERa and NF-?B was recruited to miR-31 promoter.In order to reveal the role of miR-31 in regulate mammary development, we generated the miR-31 constitutive KO mice and the miR-31cKO mice (K14-Cre:: miR-31f/f). We found a mammary phenotype of precocious alveolar differentiation in miR-31 KO mice at puberty resembling the phenotype glands in early pregnancy. During pregnancy the impaired milk production and alveogenesis were observed in miR-31 KO mice. Furthmore, the percentage of MaSCs population was significantly lower in miR-31 KO mice than that in WT by FACS analysis showed. To further determine the effect of miR-31 on repopulating capacity, we performed limiting dilution transplantation assays of MaSCs. Loss of miR-31 leads to a significantly reduced rate of successful transplantation, and less extensive mammary outgrowth.To reveal the molecular mechanism by which miR-31 functions on mammary development,we found miR-31 binding sites of GskS3?, Smad3 and Smad4. The Luciferase assay confirmed that Gsk3?, Smad3 and Smad4 were direct target genes of miR-31. Thus, wmR-31 activates Wnt signaling pathway by repressing Gsk3?, and inhibits TGF-? signaling pathways by directly regulating Smad3 and Smad4.Taken together, these results demonstrated that estrogen and progesterone responsive miR-31 promotes MaSCs expansion and prevents precocious of mammary gland through sustained activation of Wnt signaling pathway and repression of TGF-? signaling pathways.