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

We also identified and characterized the core promoter of the porcine miR-1306 gene as well as the binding regions for transcription factors such as breast cancer 1 (BRCA1), Sp1 transcription factor (SP1), and upstream transcription factor 2 (USF2) (Supplementary Figure S7) . We detected four SMAD4-binding elements (SBEs) (Figure 5A ), indicating that SMAD4 may act as a transcription factor and regulate miR-1306 transcription. To confirm this, recombinant reporter vectors containing SBEs (wild-type or mutant-type) were constructed ( Figure 5B ) and co-transfected along with SMAD4 overexpression into the porcine GCs. Results showed that the luciferase activity of plasmids with the wild-type SBE, SBE1-mut, and SBE4-mut were dramatically enhanced after SMAD4 overexpression, but that of the SBE2/3-mut construct remains the same ( Figure 5C ), suggesting that SMAD4 inhibits miR-1306 transcriptional activity via SBE2/3 motif of the core promoter. Subsequently, using a ChIP assay, we confirmed that only the SBE2/3 motif within the miR-1306 promoter could specifically bind to SMAD4 ( Figure 5D ). Collectively, these results provide compelling evidence that SMAD4 directly interacts with SBE2/3 motif and inhibits the miR-1306 gene transcription in GCs. overexpression into the porcine GCs. Results showed that the luciferase activity of plasmids with the wild-type SBE, SBE1-mut, and SBE4-mut were dramatically enhanced after SMAD4 overexpression, but that of the SBE2/3-mut construct remains the same ( Figure 5C ), suggesting that SMAD4 inhibits miR-1306 transcriptional activity via SBE2/3 motif of the core promoter. Subsequently, using a ChIP assay, we confirmed that only the SBE2/3 motif within the miR-1306 promoter could specifically bind to SMAD4 ( Figure 5D ). Collectively, these results provide compelling evidence that SMAD4 directly interacts with SBE2/3 motif and inhibits the miR-1306 gene transcription in GCs.