The Function And Mechanism Of LncRNA Silnr1 In Regulating Pluripotency Of Stem Cells And Reprogramming

Background: The discovery of i PSCs induces seminal breakthrough in the development of regenerative medicine.While reprogramming is time-consuming and low efficient,which has limited the extensive clinical application of i PSCs.As an important part of the cohesin complex,Smc1 is essential to maintain the 3 dimensional structure of the genome.The Smc1 mediated intrachromosomal loops between the enhancer and promoter of stemness genes represent a critical epigenetic step on the road to pluripotent reprogramming.To decipher the pluripotency-associated cohesin chromatin architecture,we performed RIP-seq to profile the lnc RNAs that interact with Smc1 during reprogramming.In combination with previous transcriptome sequencing result of i PSCs and fibroblasts,we identify Silnr1 as a novel reprogramming-associated lnc RNA.Silnr1 is not only a critical component of the cohesin-Smc1 complex,but is also specifically activated during the induction of pluripotency.Further experiments will explore the function and mechanism of Silnr1 in regulating pluripotency and reprogramming.Research Objectives: 1.Screen the expression of Silnr1 among different cells and tissues in variously pluripotent status,explore its association with pluripotency.2.Illustrate the function of Silnr1 in regulating pluripotency and reprogramming process.3.Identify the target gene of Silnr1 in regulating pluripotency of stem cells and reprogramming through screening its interacting network.4.Investigate the potential mechanism of Silnr1 in regulating pluripotency and reprogramming.Methods: 1.Selecting targeted lnc RNA Silnr1 through combing RIP-seq with RNA-seq.Confirming the expression of Silnr1 among different cells and tissues by RT-q PCR.Observing the expression of Silnr1 during EB body differentiation,identifying the relationship between Silnr1 and pluripotency.Confirming the subcellular location of Silnr1 with RNA-FISH.2.Constructing the plasmids for the function assay.Performing the function assay of Silnr1 through its kncokdown in i PSCs and its overexpression in fibroblasts.Detecting the expression level of stemness genes with RT-q PCR and immunoflurosence.Reprgraming assay to evaluate the influence of Silnr1 on reprogramming efficiency.3.Exploring the interaction network of Silnr1 and identifying its target gene of regulating pluripotency and reprogramming with RAT-seq.4.Exploring the potential mechanism of Silnr1 in regulating pluripoteny and reprogramming process through chromosome confirmation capture(3C)and chromatin immunoprecipitation(Ch IP).Results: 1.All together 10 lnc RNAs that were Smc1 bound as well as higher expressed in i PSCs were identified via combining RIP-Seq with RNA-Seq.We named the Smc1 best bound lnc RNA as Sinlr1 and performed further study.2.Silnr1 was expressed significantly higher in i PSCs and ESCs than in fibroblasts,non-reprogrammed cells and terminally differentiated tissues.The expression of Silnr1 owned the same trend with that of stemness genes during EB body differentiation.Silnr1 located in the nucleus.3.In loss of function assay,the expression of core stemness genes were down-regulated after Silnr1 knockdown.The sh Silnr1 i PSCs demonstrated differentiated morphology and the loss of immunostaining staining for the pluripotent marker Oct4,Sox2.Gain of function assay showed that Silnr1 could upregulate the expression level of Oct4,Sox2.Reprogramming assay showed that Silnr1 could enhance reprogramming efficiency.4.According to the RAT-seq result,Silnr1 bound to the enhancer and promoter elements of Oct4 gene.We also performed Silnr1 lnc RNA–Oct4 DNA FISH in i PSCs and found that Silnr1 signals overlapped with the Oct4 DNA signals.5.After Silnr1 knockdown,the intrachromosomal loops between Oct4 promoter and enhancer were aborted and the interaction between Oct4 gene and Smc1 was downregulated.Conclusions: 1.Using RIP-seq and RNA-seq,we identified a novel Smc1 bound chromatin lnc RNA Silnr1,which is associated with the pluripotency of stem cells.2.Silnr1 is essential for maintaining pluripotency.After sh RNA knockdown of Silnr1 in i PSCs,the expression of key pluripotent genes were down-regulated and i PSCs exited from pluripotency.3.Silnr1 could activate stemness genes,promote the pluripotency genes expression.Silnr1 could enhance the reprogramming efficiency.4.Silnr1 binds to the promoter and enhancer elements of Oct4,where it recruits Smc1 to orchestrate functional intrachromosomal loops for the establishment of pluripotency.