【作者】 刘萍；

【导师】 Miguel Angel Esteban；

【作者基本信息】 安徽大学 ， 生物化学与分子生物学， 2017， 硕士

【摘要】 转录因子介导的体细胞重编程技术不仅避免了传统干细胞研究的伦理问题;同时在疾病模型、药物筛选等方面具有广阔的应用前景。系统地研究重编程过程中的分子机制不仅有助于理解细胞命运转变的内在联系,更有助于获得临床级别的诱导多能干细胞(iPSCs)用于疾病治疗。近些年的研究表明环状RNA(circRNA)的表达呈现出细胞类型特异性且参与调控大脑神经发育、肿瘤的发生发展,暗示其具有重要的生物学功能。但circRNA在体细胞重编程和多能性中的研究还未见报道。本论文第一部分以小鼠胚胎干细胞(mESCs)和体细胞重编程为模型探索circRNA的生物学功能。首先采用环状RNA测序的方法系统的比较circRNA在胚胎干细胞和胚胎成纤维细胞(MEFs)中的表达差异;然后结合生物信息学分析和功能预测,筛选出特定的circRNA进行双向PCR测序验证;进一步针对特定的circRNA,通过siRNA敲低和过表达研究其对小鼠胚胎干细胞多能性的调控;最后在OG2MEF细胞中筛选能够调控重编程的circRNA分子。研究发现circGatad2a,circKdm2a,circMad111过表达可以稳定地促进OG2MEF细胞的重编程效率。进一步研究表明这三种环状RNAs过表达能够不同程度的促进重编程后期多能性基因(如Nanog,Pou5f1等)的表达且不依赖于自身mRNA基因的变化。本论文第二部分基于课题组已有的发现:即敲低抑制复合物NCoR/SMRT能显著提高OSKM介导的小鼠体细胞重编程效率并促进多能性基因的表达,进一步探索NCoR/SMRT抑制复合物和重编程因子间的作用关系。并以此高效重编程系统为模型,初步研究促重编程circRNA表达的差异。研究发现,重编程因子OSKM都能和NCoR/SMRT复合物相互作用,但与c-Myc的作用最强。为此我们推测NCoR/SMRT抑制复合物主要由c-Myc招募去抑制重编程后期多能性基因的激活,从而降低体细胞重编程的效率,但敲低NCoR/SMRT介导的高效重编程系统并未影响部分促重编程circRNA的表达。综上所述,本论文主要研究circRNA在小鼠胚胎干细胞和体细胞重编程中的生物学功能。通过高通量的circRNA测序和系统的功能筛选发现circRNA circGatad2a,circKdm2a-1#,circMad111可以促进体细胞重编程的效率,具体的分子机制还有待于后续研究;另一方面发现c-Myc参与招募抑制复合物NCoR/SMRT去抑制多能性基因的激活,并且敲低NCoR/SMRT介导的高效重编程系统并未影响促重编程circRNA的表达。更多还原

【Abstract】 Somatic cell reprogramming mediated by defined transcription factors avoids the ethical issues of stem cell research,and provides great application potential for disease modeling and drug screening.Systematic investigation of iPSCs reprogramming not only uncovers the intrinsic mechanism of cell fate transition but also helps to get clinical-grade iPSCs for cell therapy.Recent studies showed that circular RNAs(circRNAs)expression is cell type specific and it plays important roles in brain development and tumor progression,which suggests circRNAs have important function.While no circRNA studies have been reported either in somatic cell reprogramming or stem cell pluripotency.In the first section,we explore the biological function of circRNAs in mouse embryonic stem cells(mESCs)and somatic cell reprogramming.First,we perform high throughput circRNA sequencing in mESCs and mouse embryonic fibroblasts(MEFs)to systematically characterize circRNAs in these two typical cell types.Combine bioinformatic analysis and functional prediction,we select a panel of circRNAs for validation by using divergent PCR and Sanger sequencing.For those validated circRNAs,we study their function in mESCs by using siRNA knockdown and circRNA overexpression.Moreover,we also performed circRNA screening in OG2MEF reprogramming.Finally,we got circGatad2a,circKdm2a and circMadlll overexpression can improve reprogramming efficiency individually.Further,we found that overexpression of those circRNAs induced pluripotency genes expression at late stage while have no effects on their parent genes expression.In the second section,we explored the interaction between repressive complex NCoR/SMRT and reprogramming factors based on previous finding that NCoR/SMRT is a roadblock for mouse somatic cell reprogramming.Also,we want to study whether the highly efficient reprogramming system achieved by knocking down NCoR/SMRT will induce pro-reprogramming circRNAs expression.Our results clearly showed that NCoR/SMRT knockdown significantly increases reprogramming efficiency.Further,we identified that c-Myc is the major factor to recruit the NCoR/SMRT repressive complex and to repress the pluripotent genes activation at the late stage of reprogramming,and thus to block iPSCs generation.While the pro-reprogramming circRNAs we identified in the first section shows no obvious change in this highly efficient reprogramming system.In summary,we have studied the biological function of circRNAs in mouse embryonic stem cells and somatic cell reprogramming.Through high throughput sequencing and functional screening,we have identified several candidates including circGatad2a,circKdm2a-1#and circMadlll that can facilitate iPSCs reprogramming while the detail mechanisms are still ongoing.On the other hand,we identified that c-Myc is responsible for recruiting NCoR/SMRT repressive complex to block pluripotent genes activation.And the expression of pro-reprogramming circRNAs we identified shows no change in highly efficient reprogramming system achieved by NCoR/SMRT knockdown.更多还原