miR-1306 Mediates the Feedback Regulation of the TGF-ß/SMAD Signaling Pathway in Granulosa Cells

Yang, Liu; Du, Xing; Liu, Lu; Cao, Qiuyu; Pan, Zengxiang; Li, Qifa

Our previous study proved that miR-1306 targets the TGFBR2 3 UTR in pigs [9] . In this study, we further confirmed that TGFBR2 is a functional target of miR-1306 in porcine GCs and miR-1306 promotes GC apoptosis by impairing TGFBR2 and inactivating TGFBR2-dependent TGF-β/SMAD signaling pathway. TGFBR2 and TGFBR2-dependent TGF-β/SMAD pathway participate in GC apoptosis, as observed in several studies conducted on mammals. In pigs, for instance, TGFBR2 is a crucial repressor of GC apoptosis [9] . Furthermore, TGF-β1 could activate TGF-β/SMAD pathway and inhibit GC apoptosis, while blocking the TGF-β/SMAD signaling pathway by a specific inhibitor such as LY2157299, which can promote GC apoptosis [23, 41] . Similarly, the TGF-β/SMAD signaling pathway is also known to play an important role in GC apoptosis in mammals such as humans and rodents [42] [43] [44] [45] . While the function of miRNA-1306 has been seldom reported, studies have strongly suggested that miRNA-1306 can target and inhibit ADAM10 gene, a key gene of Alzheimer's disease (AD) [46] . Our findings define the function of miR-1306 in GC apoptosis as well as follicular atresia in mammals. The results of our study also proved that miR-1306 might serve as a molecular stimulator for female fertility. Moreover, our findings reveal the mechanism of action of miR-1306 in GCs as well as its ovarian functions. mRNAs are ubiquitous in the genome of eukaryotes and occur within intragenic regions such as introns and exons, or within intergenic regions. The proportion of intragenic miRNAs varies from approximately 33% in pigs to 55% in mouse, and most of them are intronic miRNAs, while very few are exonic miRNAs [47] . The transcriptional regulation and functions of intragenic miRNAs and their host genes has been a hot topic of research [48, 49] . Most intronic miRNAs are seen to co-express and are functionally consistent with their host genes [48, 50, 51] . A recent study showed that the transcription of intronic miRNAs does not depend on host genes and their functionally coordination is less extensive than expected [52] . However, the relationship between exonic miRNAs and their host genes still remains unknown [53] . Our study suggests that although the transcription of miR-1306, which is an exonic miRNA, is suppressed by the transcription factor SMAD4, its host gene DGCR8 is not regulated by SMAD4 in porcine GCs. Furthermore, we also demonstrated that miR-1306 has its own internal promoter that is independent of the host gene, and that SMAD4 serves as a negative regulatory factor and suppresses miR-1306 by directly binding to SBE motif within this promoter region. Therefore, our findings provide strong evidence that the exonic miR-1306 is transcribed independently of the host gene in porcine GCs. This not only clarifies the relationship between miR-1306 and its host gene DGCR8 but also lays a foundation for the further understanding of its regulatory mechanism.