SMAD7 disrupts the TGF‐β signaling pathway by influencing TGFBR1 stability and by blocking the binding of TGFBR1 to SMAD2/3. In this study, we showed that SMAD7 attenuated the TGF‐β signaling pathway in ovarian granulosa cells (GCs) by regulating TGFBR1 transcriptional activity. To function as a transcription factor, SMAD7 downregulated the mRNA levels of TGFBR1 via direct binding to the SMAD‐binding elements (SBEs) within the promoter region of pig TGFBR1. We also showed that SMAD7 enhanced porcine GC apoptosis by interrupting TGFBR1 and the TGF‐β signaling pathway. Interestingly, miR‐181b, a microRNA that is downregulated during porcine follicular atresia, was identified to be directly targeting SMAD7 at its 3′‐UTR. By inhibiting SMAD7, miR‐181b could inhibit GC apoptosis by activating the TGF‐β signaling pathway. Our findings provide new insights into the mechanisms underlying the regulation of the TGF‐β signaling pathway by SMAD7 and miR‐181b.
ABSTRACT SMARCA2, an evolutionarily conserved catalytic ATPase subunit of SWI/SNF complexes, has been implicated in development and diseases; however, its role in mammalian ovarian function and female fertility is unknown. Here, we identified and characterized the 3′-UTR of the porcine SMARCA2 gene and identified a novel adenylate number variation. Notably, this mutation was significantly associated with sow litter size traits and SMARCA2 levels, due to its influence on the stability of SMARCA2 mRNA in ovarian granulosa cells (GCs). Immunohistochemistry and functional analysis showed that SMARCA2 is involved in the regulation of follicular atresia by inhibiting GC apoptosis. In addition, miR-29c, a pro-apoptotic factor, was identified as a functional miRNA that targets SMARCA2 in GCs and mediates regulation of SMARCA2 expression via the NORFA–SMAD4 axis. Although a potential miR-29c-responsive element was identified within NORFA, negative regulation of miR-29c expression by NORFA was not due to activity as a competing endogenous RNA. In conclusion, our findings demonstrate that SMARCA2 is a candidate gene for sow litter size traits, because it regulates follicular atresia and GC apoptosis. Additionally, we have defined a novel candidate pathway for sow fertility, the NORFA–TGFBR2–SMAD4–miR-29c–SMARCA2 pathway. This article has an associated First Person interview with the first author of the paper.
Transforming growth factor-β receptor II (TGFBR2), the type II receptor of the TGF-β/SMA- and MAD-related protein (SMAD) signaling pathway, plays a crucial role in TGF-β signal transduction and is regulated by multiple factors. Nevertheless, the modulation of the non-coding RNA involved in the process of TGFBR2 expression in ovaries is not well studied. In our study, we isolated and characterized the 3′-untranslated region (UTR) of the porcine TGFBR2 gene and microRNA-1306 (miR-1306) was identified as the functional miRNA that targets TGFBR2 in porcine granulosa cells (GCs). Functional analysis showed that miR-1306 promotes apoptosis of GCs as well as attenuating the TGF-β/SMAD signaling pathway targeting and impairing TGFBR2 in GCs. Moreover, we identified the miR-1306 core promoter and found three potential SMAD4-binding elements (SBEs). Luciferase and chromatin immunoprecipitation (ChIP) assays revealed that the transcription factor SMAD4 directly binds to the miR-1306 core promoter and inhibits its transcriptional activity. Furthermore, the TGF-β/SMAD signaling pathway is modulated by SMAD4 positive feedback via inhibition of miR-1306 expression in GCs. Collectively, our findings provide evidence of an epigenetic mechanism that modulates as well as mediates the feedback regulation of the classical TGF-β/SMAD signaling pathway in GCs from porcine ovaries.
Follicular atresia occurs in every stage of ovarian development, which is relevant to female fertility. In the past decade, increasing studies have confirmed that miRNAs, a class of short non-coding RNAs, play an important role in follicular atresia by post-transcription regulation of their target genes. However, the function of miRNAs on follicular atresia initiation is unknown. In the present study, high-throughput small RNA sequencing was performed to analyze differential miRNA expression profiles between healthy (HF) follicles and early atretic (EAF) follicles. A total of 237 conserved miRNA were detected, and the miR-143 is the highest expressed in follicles. Meanwhile, we also found wide sequence variations (isomiRs) in porcine ovarian miRNA, including in 5′un-translation region, core seed sequences and 3′untranslation region. Furthermore, we identified 22 differentially expressed miRNAs in EAF groups compared to HF group, of which 3 miRNAs were upregulated, as well as 19 miRNAs were downregulated, and then the RT-PCR was performed to validate these profiles. The target genes of these differentially expressed miRNAs were predicted by using miRwalk, miRDB, and Targetscan database, respectively. Moreover, the gene ontology and KEGG pathway enrichment established that the regulating functions and signaling pathways of these miRNAs contribute to follicular atresia initiation and cell fate. In conclusion, this study provides new insights into the changes of miRNAs in early atretic follicles to demonstrate their molecular regulation in ovarian follicular atretic initiation.
Increasing evidence shows that lncRNAs, an important kind of endogenous regulator, are involved in the regulation of follicular development and female fertility, but the mechanism remain largely unknown. In this study, we found that SDNOR, a recently identified antiapoptotic lncRNA, is a potential multifunctional regulator in porcine follicular granulosa cells (GCs) through RNA-seq and multi-dimension analyses. SDNOR-mediated regulatory networks were established and identified that SOX9, a transcription factor inhibited by SDNOR, mediates SDNOR's regulation of the transcription of downstream targets. Functional analyses showed that loss of SDNOR significantly impairs GC morphology, inhibits cell proliferation and viability, reduces E2/P4 index, and suppresses the expression of crucial markers, including PCNA, Ki67, CDK2, CYP11A1, CYP19A1, and StAR. Additionally, after the detection of ROS, SOD, GSH-Px, and MDA, we found that SDNOR elevates the resistance of GCs to oxidative stress (OS) and also inhibits OS-induced apoptosis. Notably, GCs with high SDNOR levels are insensitive to oxidative stress, leading to lower apoptosis rates and higher environmental adaptability. In summary, our findings reveal the regulation of porcine GCs in response to oxidative stress from the perspective of lncRNA and demonstrate that SDNOR is an essential antioxidative lncRNA for maintaining the normal state and function of GCs.
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