circ-ANGPT1调控氧化应激诱导的猪颗粒细胞凋亡的机制

Increased follicular atresia followed by decreased reproductive capability of sows caused by oxidative stress has always been an important problem which needs to be urgently solved in the porcine production field. Recent studies have shown that circular RNA can regulate oxidative stress-induced apoptosis, however, whether it can also regulate the apoptosis of ovarian granulosa cells is still unclear. circ-ANGPT1, a novel circular RNA, was found in our previous research. The expression of circ-ANGPT1 was significantly decreased in granulosa cells from antral atretic follicles and was expected to be a key RNA regulating apoptosis induced by oxidative stress. In order to testify this hypothesis and further unravel its underlying mechanism, the porcine granulosa cells will be used as the In Vitro model. With gene knockdown and overexpression, the inhibitory effect of circ-ANGPT1 on granulosa cell apoptosis will be firstly investigated. Then using molecular techniques such as RNA immunoprecipitation, the binding proteins of circ-ANGPT1, related to the pathway of PI3K-AKT-FOXOs, will be selected. Finally, using circ-ANGPT1 knockdown combined with knockdown/overexpression of key proteins simultaneously and also ovarian oxidative stress mouse model, the effect of circ-ANGPT1 through key proteins on apoptosis of granulosa cell under the conditions of oxidative stress was further investigated. These results will not only clarify the mechanism of circ-ANGPT1 in inhibiting granulosa cell apoptosis but also can provide a scientific background for improving ovarian follicular development and increasing reproductive capability in sows under conditions of oxidative stress in the porcine production field.

氧化应激引起卵泡闭锁和母猪繁殖力下降一直是养猪生产中亟需解决的重要问题。最新研究表明：环状RNA可调控氧化应激诱导的细胞凋亡，但其能否调控卵泡颗粒细胞凋亡尚不清楚。前期通过测序，发现猪有腔闭锁卵泡颗粒细胞中显著下调的新型环状分子，circ-ANGPT1，其很可能是调控氧化应激诱导颗粒细胞凋亡的关键分子。为深入研究其调控机制，项目拟以猪颗粒细胞氧化应激模型为对象，利用干扰和过表达技术，首先明确新型环状分子对颗粒细胞凋亡的调控作用；进而利用RNA免疫共沉淀等分子技术，确定其与PI3K-AKT-FOXOs信号轴相关的结合蛋白；最后通过同时干扰环状分子和干扰/过表达关键蛋白等技术并利用小鼠卵巢氧化应激模型，揭示新型环状分子通过其下游蛋白靶点对颗粒细胞凋亡的调控机制。此结果不仅能够阐明新型环状分子调控氧化应激诱导的颗粒细胞凋亡的机制，也为改善氧化应激环境下母猪卵泡发育、提高其繁殖力提供科学理论依据。

本研究的目的是探索circRNA和lncRNA在猪卵泡闭锁中的分子机制。首先，分析健康卵泡和闭锁卵泡颗粒细胞中circRNA的分布情况，circRNA的表达分析显示共有62个circRNA表达差异显著，其中49个circRNA表达显著下调，13个circRNA表达显著上调。对筛选出差异表达的circRNA，进行GO和KEGG分析；然后通过qRT-PCR结合RNase R对差异表达circRNA进行验证，预测circRNA特定的靶向基因，后预测与卵泡闭锁的相关通路。接着，筛选了三个潜在的circRNA分子进行体外功能验证，分别为在闭锁卵泡中显著下调的circANGPT1和circANKHD1以及上调的circKIF16B；利用原代猪颗粒细胞作模型，通过一系列分子试验等确定了降低circANGPT1 和circANKHD1可促进颗粒细胞的凋亡，而过表达这两个分子则会一致颗粒细胞凋亡在颗粒细胞中具有抗凋亡作用；与之相反，过表达circKIF16B具有促进颗粒细胞凋亡的作用；通过生物信息分析及靶标关系验证发现，circ-ANKHD1可通过调控miR-27a-3p/SFRP1信号轴促进颗粒细胞增殖和抑制细胞凋亡。circKIF16B是通过ssc-miR-493-3p/ p53/PHLDA3信号通路发挥其促凋亡的作用。而circANGPT1的抗凋亡的分子作用机制还有待进一步研究中。除了circRNA，我们也利用RNA-seq获得关于早期有腔闭锁和健康有腔卵泡的猪颗粒细胞中lncRNA转录组的新信息。共鉴定出173个差异表达的lncRNAs（后续称为DE-lncRNAs）。上调的DE-lncRNAs的潜在mRNA靶点主要集中在凋亡相关通路中，而下调的DE-lncRNAs的靶点基因主要集中在代谢和卵巢类固醇生成通路中。线性回归分析显示上调的DE-lncRNAs表达与凋亡相关基因显著相关。在预测了miRNA结合位点的lncRNA中，NOVEL_00001850是表达下调幅度最大的DE-lncRNA。qRT-PCR和线性回归分析证实了其潜在靶基因CYP19A1的表达和相关性，CYP19A1是参与雌二醇合成的关键基因。我们的结果表明，lncRNAs可能参与颗粒细胞凋亡，从而导致有腔卵泡闭锁。.综上所述，环状RNA和lncRNA通过多种分子机制共同调控猪颗粒细胞的凋亡及有腔卵泡的闭锁过程。