Mechanism Of LncRNA Oplr16 Controls Stem Cell Fate And Reprogramming Through Coordinating Chromosomal Looping

Background:Induced pluripotent stem cells(iPSC)are formed by reprogramming somatic cells with specific factors.Because of its ability to self-renew and differentiate into three germ layers,it has application prospects for establishing disease models,drug screening,and personalized regenerative medicine.At present,iPSC-derived NK cells and iPSC-based tumor vaccines are hotspots.However,the iPSC reprogramming process has a technical bottleneck of low-efficiency and time consuming.Transcription factor c-Myc has potential carcinogenic risks,and the usage of viral vectors may lead to genomic instability.These disadvantages greatly limit iPSC's application to the clinic.Scientists have tried to improve iPSC reprogramming by modifying the Wnt signaling pathway,the ERK / MAPK signaling pathway,and usage of small chemical compounds.However,the results obtained so far are unsatisfactory.Only by deeply studying the iPSC reprogramming mechanism can we break the technical bottleneck,optimize the iPSC,and promote its clinical transformation.iPSC undergoes unique epigenetic changes during reprogramming,including DNA methylation,histone modification,and formation of chromosomal looping structures.Recent studies have found that lncRNA exhibits strong specificity in cells,plays an important role in many pathological and physiological processes,and also participates in regulating pluripotency and differentiation of stem cells.LncRNA regulates epigenetics by combining with chromatin DNA,recruiting regulatory factors,and changing DNA epigenetic modifications.Objectives:Finding out lncRNAs which are related to stem cell pluripotency,studying their molecular mechanism regulating stem cell pluripotency,and exploring clinical transformation through lncRNAs as a medium to achieve manipulation and control of cell fate.Methods:Scr eening lncRNAs: Firstly combined "chromatin RNA in situ reverse transcription-associated trap sequencing(CRIST-Seq)" and different expression between FBC and iPSC RNA-Seq technology to screen pluripotency-related lncRNAs that bound to the pluripotency gene Oct4 promoter.The subcellular localization of lncRNA was determined by nuclear and cytoplasm sepration experiment and RNA FISH.LncRNA function study: The embryonic body formation was used to observe the relationship between lncRNA and cell pluripotency.The shRNA knockdown was used to detect the expression levels of lncRNA and pluripotency genes Oct4,Sox2,Nanog,and to observe the changes of cell morphology.An overexpression technology was used to check out whether in vitro and in vivo overexpression of lncRNA promoted pluripotency gene expression.Through induction reprogramming experiments,it was clarified whether overexpression of lncRNA improved reprogramming efficiency.LncRNA mechanism study: Reverse transcription associated trap sequencing(RAT-Seq)was used to determine the genomewide target network with which the lncRNA interacts,and RNA-DNA FISH was used to determine the spatial relationship between lncRNA and target gene.The 3C technique was used to determine the effect of lncRNA on the structure of chromosomal looping,then RIP and RNA binding techniques were used to determine whether lncRNA can recruit SMC1 and specific binding fragments.The methylation level of the promoterregion of the pluripotent gene of fibroblasts overexpressing lncRNA was detected,RIP technology was used to determine whether lncRNA recruited demethylase.RNA mapping technology was used to determine the specific lncRNA and demethylase binding site.Results:LncRNA scr eening: By combining CRIST-Seq and RNA-Seq,we screend out lncRNAs that were highly expressed in iPSC but low in FBC.We found out lncRNA Oplr16,a new long non-coding RNA had not been reported so far,meets both of the above criterias.Oplr16 is 629 bp in length and is located on mouse chromosome 17.Oplr16 was specifically expressed in stem cells,and was barely detected in other tissues.Nuclear and cytoplasm fractionation assay and RNA FISH showed that Oplr16 was mainly distributed in the nucleus.Functional study: The results of embryoid body formation showd that Oplr16,paralleled with important pluripotency genes such as Oct4,Sox2,and Nanog,was decreased in expression during cell differentiation.Using shRNA to knock down Oplr16,the expression of Oct4,Sox2,and Nanog was also significantly reduced following the decreasion of Oplr16.The morphology of iPSC that were successfully transfected with shOplr16 showed characterization of differentiation,and lost pluripotency marker SOX2.The morphology of iPSC transfected with sh-CT,however,remained normal,and pluripotency marker SOX2 expressed well.The pOct4-luciferase assay showed that the luciferase activity of the Oplr16 overexpression group was nearly 8 times higher than that of the blank plasmid group and the random lncRNA plasmid group.The level of Oct4 transcription in FBC transfected with the Oplr16 overexpression plasmid increased by more than4-fold.After transfection of the Oplr16 overexpression plasmid,the DOX-OSKM-MEFs exhibited high reprogramming efficicency with more iPSC clones.Mechanism study: RAT results showed that Oplr16 was significantly enriched in the Oct4 promoter region.Quantitative PCR further confirmed the enrichement of Oplr16 in the Oct4 promoter.RNA-DNA FISH found that Oplr16 was spatially adjacent to Oct4.3C technology was used to determine the role of Oplr16 in maintaining the chromosomal looping structure of Oct4.In iPSC transfected with shOplr16 plasmid,Oct4 chromosomal looping structure was significantly reduced.The SMC1 RIP technology showed that Oplr16 interacted with SMC1,and the SMC1-RNA Binding test suggested that the 3' end of Oplr16 interacted with SMC1.In FBCs transfected with the Oplr16 overexpression plasmid,the rate of demethylation of the CpG islands in the Oct4 promoter region was significantly higher than that of the blank control plasmid and the random control plasmid group.After transfection of the Oplr16 overexpression plasmid,the expression level of Tet2 increased significantly.TET2 RIP technology detected the interaction between Oplr16 and DNA demethylase TET2.The RNA mapping technique suggested that the 3' end of Oplr16 binds to TET2.Overexpression of the 3'-end deleted Oplr16 abolished the idnuced demethylation of CpG islands in the Oct4 promoter.Conclusions:Oplr16 is a new pluripotent lncRNA,and is specifically and highly expressed in iPSC.After knocking down Oplr16,the morphology of iPSC changes,showing typical differentiation.The expression of pluripotency-associated genes are down-regulated accordingly.Overexpressing Opr16 improves reprogramming efficiency.Oplr16 regulated stem cell pluripotency through multiple mechanisims,including activating the endogenous pluripotency gene Oct4,interacting SMC1 protein to promote the formation of chromosomal looping structure,promoting Tet2 expression and recruiting TET2 protein to induce DNA demethylation.