Laboratory of Statistical Genetics and Epigenetics, College of Animal Science and Technology, Nanjing Agricultural University, Nanjing, China.
Cell Prolif. 2023 Jan;56(1):e13336. doi: 10.1111/cpr.13336. Epub 2022 Sep 20.
Transforming growth factor β1 (TGF-β1), an essential cytokine belongs to TGF-β superfamily, is crucial for female fertility. Increasing evidence show that long noncoding RNAs (lncRNAs) influence the state of granulosa cells (GCs). This study aimed to detect the effects of TGF-β1 on the lncRNA transcriptome, and investigate whether lncRNAs mediate the functions of TGF-β1 in GCs.
RNA-seq and bioinformatics analyses were performed to identify and characterize the differentially expressed lncRNAs (DElncRNAs). The regulatory mechanism of TGF-β1 to lncRNA transcriptome was analyzed by chromatin immunoprecipitation. The effects of lncRNAs on the antiapoptotic and proproliferative functions of TGF-β1 were examined by morphological analysis, fluorescence-activated cell sorting, Cell Counting Kit-8, and Western blot.
A total of 72 DElncRNAs highly sensitive to TGF-β1 were identified with the criteria of |log (fold chage)| ≥ 3 and false discovery rate < 0.05. Functional assessment showed that DElncRNAs were enriched in TGF-β, nuclear factor kappa B, p53, and Hippo pathways which are crucial for the normal state and function of GCs. Importantly, SMAD4 is essential for the regulation of TGF-β1 to lncRNA transcriptome. In vitro studies confirmed that TGF-β1 induced TEX14-IT1 transcription in a SMAD4-dependent manner, and TEX14-IT1 mediated the antiapoptotic and proproliferative effects of TGF-β1 in GCs.
Our findings demonstrate that TGF-β1 alters lncRNA transcriptome in a SMAD4-dependent manner, and highlight that lncRNAs mediate the functions of TGF-β1 in GCs, which contribute to a better understanding of the epigenetic regulation of female fertility.
转化生长因子β1（TGF-β1）是转化生长因子-β超家族中的一种重要细胞因子，对女性生育能力至关重要。越来越多的证据表明，长非编码 RNA（lncRNA）影响颗粒细胞（GCs）的状态。本研究旨在检测 TGF-β1 对 lncRNA 转录组的影响，并探讨 lncRNA 是否介导 TGF-β1 在 GCs 中的功能。
采用 RNA-seq 和生物信息学分析鉴定和描述差异表达的 lncRNA（DElncRNA）。通过染色质免疫沉淀分析 TGF-β1 对 lncRNA 转录组的调控机制。通过形态分析、荧光激活细胞分选、细胞计数试剂盒-8 和 Western blot 检测 lncRNA 对 TGF-β1 抗凋亡和促增殖功能的影响。
根据 |log（fold chage）|≥3 和错误发现率<0.05 的标准，共鉴定出 72 个对 TGF-β1 高度敏感的 DElncRNA。功能评估表明，DElncRNA 富集在 TGF-β、核因子 kappa B、p53 和 Hippo 通路中，这些通路对 GCs 的正常状态和功能至关重要。重要的是，SMAD4 是 TGF-β1 调节 lncRNA 转录组所必需的。体外研究证实，TGF-β1 以 SMAD4 依赖的方式诱导 TEX14-IT1 转录，TEX14-IT1 介导 TGF-β1 在 GCs 中的抗凋亡和促增殖作用。
我们的研究结果表明，TGF-β1 以 SMAD4 依赖的方式改变 lncRNA 转录组，并强调 lncRNA 介导 TGF-β1 在 GCs 中的功能，这有助于更好地理解女性生育能力的表观遗传调控。
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