Lefty1对小鼠胚胎干细胞分化过程的影响

The Role of Lefty1 in the Process of Mouse Embryonic Stem Cell Differentiation

目的:研究Lefty1在小鼠胚胎干细胞分化过程中的作用。方法:根据染色质免疫沉淀测序结果,在临近Lefty1转录起始位点以及与之相距10 kb的上游区域有TGF-β信号通路Smad2/3蛋白的四个结合区域,通过CRISPR/Cas9方法获得四个区域敲除的单克隆细胞,利用荧光实时定量PCR(qRT-PCR)检测各细胞中Lefty1的转录水平,并用TGF-β信号通路的激活剂AC和抑制剂SB分别处理敲除的细胞,检测其对TGF-β信号的响应,最后通过胚状体形成实验,检测敲除细胞系在中内胚层分化过程中的标志分子Gsc和Mixl1的表达。结果:利用CRISPR/Cas9方法成功获得不同区域敲除的单克隆细胞,与野生型E14细胞相比,四种区域敲除的细胞中Lefty1 RNA含量明显降低,并且在干细胞状态和分化状态下,敲除细胞系对TGF-β信号的响应减弱。在胚状体形成的实验中,与野生型E14细胞相比,敲除细胞系在分化过程中Lefty1表达的基础水平明显降低,中内胚层分化的标志分子Gsc和Mixl1转录水平也明显下降。结论:在胚胎干细胞中,Lefty1转录起始位点附近以及上游10 kb的这四段区域通过TGF-β信号通路对Lefty1的转录发挥调控作用,从而影响中内胚层分化过程中的标志分子Gsc和Mixl1的表达。

Objective: To study the role of Lefty1 in the process of mouse embryonic stem cell differentiation. Methods: According to the chromatin immunoprecipitation sequencing results, there are four binding regions of Smad2/3 protein near above and 10 kb upstream from Lefty1 transcription starting site. To achieve knock out cell lines of four different regions by the CRISPR/Cas9 system, the transcription level of Lefty1 was detected by using real-time quantitative PCR. The knockout cells were treated with the TGF-β activator AC and inhibitor SB, to detect TGF-β signal response. Then, the expression level of mesendoderm markers Gsc and Mixl1 was detected during embryoid body formation in knockout cell lines. Results: The different four regions were knocked out by the CRISPR/Cas9 system. Compared with the wild type, the transcription level of Lefty1 RNA in knockout cell lines was significantly decreased, and the TGF-β signal response of knockout cell lines was lower in ES state and EB state. Compared with the wild type, the basal level of Lefty1 expression in the knockout cell lines was significantly reduced, and the transcription level of mesendoderm markers Gsc and Mixl1 was also decreased significantly during embryoid body formation. Conclusions: The regions near above and 10 kb upstream from Lefty1 transcription start site regulate the transcription process of Lefty1 through the TGF-β pathway-dependent manner, which further regulates the expression of mesendoderm markers Gsc and Mixl1.