环状RNAs在干细胞多能性和重编程中的功能研究

发布时间：2018-08-05 16:48

【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。
[Abstract]:Transcription factor-mediated somatic cell reprogramming not only avoids the ethical problems of traditional stem cell research, but also has a broad application prospect in disease model and drug screening. A systematic study of the molecular mechanisms in the process of reprogramming not only helps to understand the intrinsic relationship of cell fate transition, but also helps to obtain clinically induced pluripotent stem cells (iPSCs) for disease treatment. Recent studies have shown that the expression of cyclic RNA (circRNA) is cell-specific and involved in the regulation of brain neurogenesis and tumor development, suggesting that it has important biological functions. However, the study of circRNA in somatic reprogramming and pluripotency has not been reported. In the first part of this thesis, (mESCs) and somatic cell reprogramming of mouse embryonic stem cells were used as models to explore the biological function of circRNA. The differential expression of circRNA in (MEFs) of embryonic stem cells and embryonic fibroblasts was systematically compared by cyclic RNA sequencing, and then specific circRNA was selected for bidirectional PCR sequencing by bioinformatics analysis and function prediction. Furthermore, the regulation of mouse embryonic stem cell pluripotency was studied by siRNA knockdown and overexpression for specific circRNAs. Finally, the reprogrammed circRNA molecules were screened in OG2MEF cells. It has been found that overexpression of circGatad2atio circKdm2aSc Mad111 can stably promote the reprogramming efficiency of OG2MEF cells. Further studies have shown that these three cyclic RNAs overexpression can promote the expression of polymorphic genes (such as Nanog-Pou5f1, etc.) in the later stage of reprogramming, and do not depend on the changes of their own mRNA genes. The second part of this thesis is based on the findings of the research group: knock down inhibition complex NCoR/SMRT can significantly improve the efficiency of OSKM mediated somatic cell reprogramming and promote the expression of pluripotent genes in mice. To further explore the interaction between NCoR/SMRT inhibitory complex and reprogramming factor. Based on the model of high efficiency reprogramming system, the difference of circRNA expression in accelerated reprogramming is preliminarily studied. It was found that the reprogramming factor OSKM could interact with NCoR/SMRT complex, but had the strongest effect with c-Myc. Therefore, we speculate that the NCoR/SMRT inhibitory complex is mainly recruited by c-Myc to suppress the activation of pluripotent genes in the late stage of reprogramming, thus reducing the efficiency of somatic reprogramming. But knock-down NCoR/SMRT-mediated high-efficiency reprogramming system did not affect the expression of partial re-programming circRNA. To sum up, the biological function of circRNA in mouse embryonic stem cell and somatic cell reprogramming was studied. Through high-throughput circRNA sequencing and systematic functional screening, it was found that circRNA circGatad2ahongcircKdm2a-#1 circMad111 could promote the efficiency of somatic cell reprogramming, and the specific molecular mechanism needed to be further studied. On the other hand, it was found that c-Myc was involved in the recruitment of inhibition complex NCoR/SMRT to inhibit the activation of pluripotent genes, and knockdown of NCoR/SMRT mediated high efficiency reprogramming system did not affect the expression of circRNA.
【学位授予单位】：安徽大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q78

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