LncRNA-066/236簇介导TGF-β信号通路调控猪卵泡颗粒细胞中Wnt信号通路分子机制的探究

TGF-β signaling pathway has been proved to play a critical role in regulating sow reproductive performance, follicular atresia and granulosa cell (GC) apoptosis. After preliminary study, applicant found that abrogation of SMAD4, the core member of TGF-β signaling pathway, could suppress the expression of FZD4 which is a important receptor of Wnt signaling pathway in porcine GCs with an unknown mechanism. SMAD4-dependent lncRNA-miRNA-mRNA interaction network was built and showed that lncRNA-066/236 cluster with miR-29c and FZD4 could form a regulatory pathway which was further proved to be involved in regulating porcine GC apoptosis. Based on these findings, we speculate that TGF-β signaling pathway regulates Wnt signaling pathway through lncRNA-066/236 cluster-miR-29c-FZD4 axis and affects porcine GC apoptosis. To address this, the project aims to investigate the formation mechanism of SMAD4-lncRNA-066/236 cluster axis, lncRNA-066/236 cluster-miR-29c axis and miR-29c-FZD4 axis and analyze the mechanism for lncRNA-066/236 cluster mediates TGF-β signaling pathway regulation to Wnt signaling pathway. This study will provide a theoretical basis for revealing the mechanism of TGF-β signaling pathway modulating follicular atresia and improving sow fertility.

TGF-β信号通路是影响母猪繁殖性能、卵泡闭锁与颗粒细胞凋亡的关键信号通路。申请人前期研究发现敲减TGF-β通路核心成员SMAD4可抑制猪卵泡颗粒细胞中Wnt信号通路关键受体FZD4的表达，但机制尚不清楚。依赖SMAD4的lncRNA-miRNA-mRNA互作网络显示lncRNA-066/236簇可与miR-29c、FZD4形成调控通路，并证实此通路参与调控猪卵泡颗粒细胞凋亡。因此，我们猜想TGF-β信号通路可能通过上述调控轴影响Wnt信号通路，进而调控猪卵泡颗粒细胞凋亡。为了验证此假设，本项目拟分析SMAD4-lncRNA-066/236簇轴、lncRNA-066/236簇-miR-29c轴、miR-29c-FZD4轴的形成与互作机制，解析TGF-β信号通路通过lncRNA-066/236簇调控Wnt信号通路的机制，为揭示TGF-β通路调控猪卵泡闭锁的机制、提高母猪繁殖力提供依据。

TGF-β信号通路对于维持卵巢颗粒细胞正常状态和功能、卵泡发育及母猪繁殖力至关重要。申请人前期发现TGF-β信号通路核心分子SMAD4是猪卵巢颗粒细胞中Wnt信号通路核心受体FZD4的诱导因子，结合高通量测序和生物信息学分析发现猪卵巢组织特异性高表达lncRNA簇（lncRNA-066/236）可能参与上述调控过程，因此推测TGF-β信号通路通过SMAD4-lncRNA簇-FZD4轴调控Wnt信号通路并影响猪卵巢颗粒细胞状态和功能。本项目首先探究了TGF-β1对猪卵巢颗粒细胞lncRNA转录组的影响，并通过ChIP试验证实依赖SMAD4核转录活性是TGF-β信号通路调控基因转录的普遍机制。进一步对lncRNA-066/236簇进行分析发现SMAD4作为转录激活因子促进两者在颗粒细胞中转录；其次，lncRNA-066（SDNOR）通过吸附内源性miR-29c解除后者对FZD4的抑制作用，进而促进Wnt信号通路活性并抑制颗粒细胞凋亡；第三，敲减lncRNA-236（TEX14-IT1）严重损伤TGF-β1在颗粒细胞中的正常功能，包括促增殖和抑凋亡等，证实lncRNA作为表观调控因子对于TGF-β信号通路功能的完整性至关重要；最后，SMAD4作为转录激活因子促进上游受体转录，首次证实SMAD4是TGF-β家族信号通路的直接反馈调控因子。本项目揭示了TGF-β信号通路调控靶基因转录的普遍机制，丰富并拓展了TGF-β信号通路介导的表观调控机制和调控网络，研究结果对进一步理解母猪卵泡发育和繁殖性状早期形成的分子机制、提高母猪繁殖力均具有一定意义。