人早期胚胎与胚胎干细胞中RNF126介导p21等泛素化调控细胞周期的分子机制研究

Early embryo development arrest is a thorny problem in assisted reproductive treatment, and the molecular mechanisms need to be elucidated. Control of the cell cycle is the basis for normal embryo development. p21 is the main cyclin dependent kinase inhibitor in cell cycle, with a short half-life in various tissues, and therefore the degradation of p21 protein is particularly important for its normal functioning. The ubiquitin-proteasome system is the major way of protein degradation in eukaryotic cells, and our previous study discovered that RNF126 is a novel ubiquitin ligase by siRNA library screening, which could mediate the ubiquitination of p21, promote p21 degradation in G1 phase, and thus regulates cell proliferation. We confirmed the expression of RNF126 in human ovaries, which is relatively high expressed from zygote to blastocyst as detected by single-cell sequencing. However, the RNA level of RNF126 is significantly decreased in embryos blocked at 2-cell stage, which is opposite to the expression trend of p21. The current study will further investigate the molecular mechanisms of RNF126 mediated ubiquitination of p21 in regulating cell cycle of human early embryos by gene silencing, overexpression, ubiquitination analysis, accompanied by evaluating the fertility levels of conditional RNF126 knockout mice and other methods. In addition, the interaction study and functional analysis of relevant proteins will be carried out for exploring RNF126 novel substrates in embryonic development process, and thus provide potential drug targets and therapeutic strategies for reproductive medicine.

早期胚胎发育阻滞是辅助生殖治疗中的棘手难题，但其分子机制有待阐明。细胞周期调控是保证胚胎正常发育的基础，而p21是细胞周期中主要的蛋白依赖性激酶抑制剂，其半衰期较短，因此蛋白降解对其功能的正常发挥尤为重要。泛素-蛋白酶体系统是真核细胞蛋白降解的主要途径，我们前期研究发现并证明RNF126是一个新的泛素连接酶，能够介导p21泛素化并促进其在G1期降解，从而调控细胞增殖，还发现RNF126在人卵巢中有表达，并且RNF126在人受精卵直至囊胚期中均有较高表达水平，而在发育阻滞于2细胞期的人胚胎中RNA水平显著降低，与p21表达趋势相反。本课题将通过基因沉默与过表达、泛素化分析，结合条件性RNF126敲除小鼠生育力水平的变化等，阐明RNF126介导p21等泛素化调节人胚胎发育的分子机制，了解胚胎发育阻滞原因，并将探索RNF126新的关键底物，为生殖医学提供潜在药物靶点与治疗策略。

配子和早期胚胎发育阻滞是辅助生殖治疗中的棘手难题，其分子机制有待阐明。蛋白功能的正常是保证机体正常生理功能的基础，而蛋白降解对维持蛋白质稳态，从而保证其功能的正常发挥尤为重要。泛素-蛋白酶体系统是真核细胞蛋白降解的主要途径，我们前期研究发现并证明RNF126是一个新的泛素连接酶，能够介导p21泛素化并促进其在G1期降解，从而调控细胞增殖。目前的研究主要围绕RNF126在配子和早期胚胎发育中作用的分子机制，研究内容包括两个方面。其一是RNF126在卵母细胞、早期胚胎和胚胎干细胞中的分子调节机制。研究发现RNF126在人卵巢中有表达，并且RNF126在人受精卵直至囊胚期中均有较高表达水平，而在发育阻滞于2细胞期的人胚胎中RNA水平显著降低，与p21表达趋势相反。RNF126在小鼠早期胚胎中的蛋白定位表达于胞浆中，通过显微注射的方法向合子期小鼠胚胎内注PMO-Rnf126，体外培养至第 5-6天，结果提示四细胞与八细胞期，RNF126沉默后胚胎发育受限。另一方面，精母细胞减数分裂是精子形成的核心过程。在减数分裂前期I，XY染色体上的非同源区域发生性染色体沉默（MSCI）这一重要生物学事件。ATR被募集到覆盖有RPA的XY非同源区域，在MSCI激活中起中心作用，但MSCI启动机制尚未阐明。我们研究发现RNF126在人与小鼠睾丸中均呈特异性高表达，RNF126敲除小鼠睾丸体积与生育能力明显低于野生型，且精子发生阻滞于减数分裂前期I。RNF126能够泛素化MRN复合体，影响RPA介导的ATR活化及其效应激酶CHK1的磷酸化水平等。无精症患者测序结果显示，RNF126基因的3个外显子出现高频率的特定点变异。综上，项目研究结果进一步明确了RNF126调节人早期胚胎发育的分子机制，并探索了RNF126在精子发生过程中新的关键底物，为生殖医学提供潜在药物靶点与治疗策略。