The Role Of Circular RNAs In Stem Cell Pluripotency And Reprogramming

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.