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

To characterize the 3 regulatory region of the porcine TGFBR2 gene, we isolated its 3 -UTR sequence by using a RACE assay and found only one clear band, approximately 3400 bp in length (Supplementary Figure S1 ). Clone sequencing and sequence alignment showed that the full length 3 -UTR of the porcine TGFBR2 gene was 2381 bp, with 94.73% nucleotide identity with the pig genome sequence (GenBank ID: XM_021071493.1), excluding 24 bp of polyA tail (PAT) sequence (Supplementary Figure S2 ). Porcine TGFBR2 3 -UTR is highly consistent with that found in mammals, such as humans (79.79%, GenBank ID: NM_001024847.2), cattle (60.47%, GenBank ID: NM_001159566), and sheep (27.55%, GenBank ID: XM_012099307). The polyA signal (PAS) (AAUAAA) is located 28 nucleotides before the PAT sequence. In addition, this region contains several classic elements, like GU-Rich Element (GRE) (UUGUU) and AU-Rich Element (ARE) (AUUUA), and a CAA Repeat ((CAA) 6 ) (Supplementary Figure S2) . Moreover, the miRNA response elements (MREs) for 160 candidate miRNAs were identified on the 3 -UTR of porcine TGFBR2 mRNA (Supplementary Table S5) using an online program, RNAhybrid. Among them, miR-1306 not only had the highest binding capacity with the porcine TGFBR2 3 -UTR but was also observed to target porcine TGFBR2 gene [9] . Therefore, we chose miR-1306 for further studies.