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

The above results prove that TGFBR2 is a functional target of miR-1306 and SMAD4 directly inhibits miR-1306 transcription. Therefore, miR-1306 expression may be mediated through SMAD4 feedback regulation of TGFBR2. To further confirm this, we co-transfected miR-1306 inhibitor with SMAD4-siRNA and miR-1306 mimics with pcDNA3.1-SMAD4 into the GCs. After detection, we found that the knockdown of SMAD4 dramatically suppressed miR-1306 inhibitor-induced increase in TGFBR2 protein expression ( Figure 6A ). In contrast, the overexpression of SMAD4 rescued the miR-1306-induced decrease in TGFBR2 protein expression ( Figure 6B ), indicating that SMAD4 feedback activates TGFBR2 by decreasing miR-1306. Further, we tested whether miR-1306 expression levels are affected by the TGF-β/SMAD signaling pathway. Our results showed that the miR-1306 levels sharply reduced in the GCs treated with 20 ng/mL of TGF-β1 (TGF-β/SMAD signaling pathway activation) ( Figure 6C ). Therefore, miR-1306 can interact with SMAD4 and TGFBR2, which further participates in SMAD4-mediated positive feedback regulation of the classical TGF-β/SMAD signaling pathway in GCs.