长链非编码RNA lncPrep+96kb在Notch信号调控颗粒细胞分化中的作用研究

Notch signaling defines an evolutionary ancient cell interaction. In our previous study, we reported that Notch signaling was involved in follicle growth through regulating granulosa cell proliferation. In present study, we found that blocking of Notch signaling upregulated FSHR expression and overexpression of Notch2 inhibited the expression of FSHR. LncPrept+96kb is one new long non-coding RNA, which is highly expressed in granulosa cells. We found that activation of Notch signaling increased its expression in granulosa cells. RNA immunoprecipitation enriched lncPrept+96kb binding protein: USF1 and USF2, which are two important transcription factors of regulating FSHR transcription. Based on these results, we hypothesize that lncPrept+96kb is involved in FSHR expression regulated by Notch signaling. To test the hypothesis, we will employ Notch signaling knockout mice and granulosa cell culture in vitro to investigate the influence of Notch signaling on FSHR and lncPrept+96kb expression and explore the function and mechanism of lncPrept+96kb on FSHR expression regulated by Notch signaling. Completion of this study will be instrumental to understand the mechanism of Notch signaling on granulosa cell differentiation and provide novel insight for follicle development.

Notch通路是非常保守的信号传导途径，我们前期报道其通过调控颗粒细胞增殖影响卵泡发育；目前预实验发现，Notch通路抑制剂显著上调颗粒细胞FSHR水平，过表达Notch2则抑制FSHR表达。IncPrept+96kb是最新报道颗粒细胞高表达的长链非编码RNA,但作用尚不清楚。我们发现激活Notch通路显著上调了IncPrept+96kb表达；RNA免疫共沉淀发现lncPrept+96kb与调控FSHR的转录因子(USF1和USF2)直接结合。因此我们假设lncPrept+96kb在Notch调控颗粒细胞FSHR中起重要作用。本课题将通过Notch通路敲除小鼠模型和颗粒细胞培养观察Notch通路对颗粒细胞分化和lncPrep+96kb的影响，观察lncPrep+96kb在Notch通路调控FSHR中的作用及机理。该研究有助于阐明Notch调控颗粒细胞分化的机制，为卵泡发育机理提供实验依据。

Notch信号通路是非常保守的信号传导途径，我们前期报道了其通过调控颗粒细胞增殖影响卵泡发育；本课题重点研究Notch信号通路对颗粒细胞分化的影响及机制。我们发现Notch信号抑制了颗粒细胞雌孕激素的分泌，抑制了FSHR表达，抑制了激素合成关键酶（芳香化酶，P450scc和3β-HSD）和关键转录因子（SF1，GATA4，GATA6和Wt1）表达。lncPrept+96kb是最新报道的长链非编码RNA，我们通过原位杂交显示其时空特异地表达于小鼠卵巢颗粒细胞，通过过表达和敲除颗粒细胞lncPrept+96kb，我们发现lncPrept+96kb明显抑制了雌激素分泌和芳香化酶表达，也抑制了FSHR表达，但对孕激素没有明显影响；进一步研究发现激活Notch通路显著上调了lncPrept+96kb表达，lncPrept+96kb过表达可以部分逆转Notch信号抑制对颗粒细胞雌激素合成和FSHR表达的影响，提示lncPrept+96kb介导了Notch信号通路对颗粒细胞分化的影响；同时我们也发现IL6介导了Notch信号对孕激素合成的影响。深入研究lncPrept+96kb调控雌激素合成的机制，我们通过RNA免疫共沉淀发现lncPrept+96kb特异性结合EDF1并促进了EDF1的核质移位，EDF1为激素合成关键转录因子SF1等的转录共激活因子。CatRAPID预测lncPrept+96kb 1979-2077位点和603-690位点与EDF1高度结合，突变这两个位点后发现1979-2077位点突变逆转了lncPrept+96kb对雌激素分泌和芳香化酶表达的作用， 603-690位点没有明显变化。该研究详细探究了Notch信号通路对颗粒细胞分化的影响及机制，特别是深入阐明了lncPrept+96kb介导Notch信号对雌激素合成的详细机制，为卵泡发育机理提供实验依据。