Fig 2: ESR2 negatively regulates miR-135a expression via binding to miR-135a promoter region. (A) Luciferase activity assay of a series of truncated miR-135a promoters in mGCs. (B) Schematic diagram of miR-135a promoter. (C) Site-directed mutation analysis of the miR-135a promoter using luciferase activity assays. Two intact ESR2 binding sites were indicated by empty boxes, respectively. The filled boxes showed the corresponding mutations. (D) ChIP analyses of ESR2 binding sites in miR-135a promoter region followed by qPCR. mGCs expressing N-HA were assayed as a control to exclude the possibility of nonspecific binding of anti-HA. (E) Luciferase activity of miR-135a promoter was downregulated in Esr2 overexpressed mGCs. (F) Relative level of miR-135a in Esr2 overexpressed mGCs. (G) Relative level of miR-135a, Tgfbr1 and Ccnd2 expression in estrogen-treated mGCs. The means ± SD were calculated from three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001.

Fig 3: Tgfbr1 and Ccnd2 are the targets of miR-135a. Targetscan software showed the predicted miR-135a binding site in (A) Tgfbr1 and (B) Ccnd2 3′UTRs. The wild type (WT) and mutational (MUT) 3′UTRs of (C) Tgfbr1 and (D) Ccnd2 mRNAs were shown in mGCs. The mutant bases in the 3’ UTR of target genes were shown with red font. The luciferase activity of recombinant (E) Tgfbr1 and (F) Ccnd2 3′UTRs was determined in miR-135a overexpressed mGCs. NS, none significant difference. Relative mRNA level of (G) Tgfbr1 and (H) Ccnd2 was shown in miR-135 overexpressed and inhibited mGCs. Western blot analysis of (I) Tgfbr1 and (J) Ccnd2 was shown in miR-135a overexpressed and inhibited mGCs. The relative quantification of (K) TGFBR1 and (L) CCND2 protein levels was shown in the bar graph. Data were presented as mean ± SD (n = 3). * p < 0.05, ** p < 0.01.

Fig 4: A schematic model for miR-135a regulating granulosa cell growth. The schematic diagram depicting the mechanism that miR-135a regulates murine granulosa cell growth. First, miR-135a binds to 3′UTR of Tgfbr1 and Ccnd2 to repress their expression. Then, downregulated TGFBR1 fails to facilitate nuclear distribution of SMAD3, which could not enhance Ccnd2 expression. Finally, ESR2 functions as a transcription factor to directly bind to the miR-135a promoter region, decreases the transcriptional activity of miR-135a, and eventually suppresses miR-135a-mediated mGC growth suppression.

Fig 5: Tgfbr1 enhances SMAD3-mediated Ccnd2 promoter activity. (A) Western blot analysis of p-SMAD3 protein level in Tgfbr1 overexpressed mGCs. The relative quantification of p-SMAD3 protein level in Tgfbr1 overexpressed mGCs was shown in the right panel. (B) Western blot analysis of p-SMAD3 protein level in mGCs transfected with miR-135a mimics. The relative quantification of p-SMAD3 protein level in mGCs transfected with miR-135a mimics was shown in the right panel. (C) The subcellular distribution of SMAD3 was detected by immunofluorescence assay in mGCs treated with Tgfbr1 overexpression or miR-135a mimics. Scale bar: 20 μm. (D) ChIP analyses of SMAD3 binding sites in Ccnd2 promoter region followed by qPCR. (E) Relative mRNA level of Ccnd2 in Tgfbr1 overexpressed mGCs. (F) Western blot analysis of CCND2 protein level in Tgfbr1 overexpressed mGCs. The means ± SD were calculated from three independent experiments. * p < 0.05, ** p < 0.01.