Role And Mechanism Of LncRNA Peln1 In Regulating Pluripotency Exit Of Stem Cells

Background and objectives:Stem cell research is the frontier fields of biomedical research,has a broad application prospect.Induced pluripotent stem cells(iPSCs)have a more broad application prospect,because they can be obtained by somatic reprogramming,which avoids the ethical controversy in the application of embryonic pluripotent stem cells(ESCs).However,due to the lack of in-depth understanding of the formation mechanism of iPSCs,the problem of how to improve the iPSCs induction efficiency and precisely regulate the committed differentiation of cells has become a bottleneck for the application of this technology.There is increasing evidence that long non-coding RNA(lncRNA)plays an important role in many physiological and pathological activities.However,exactly which lncRNAs play a role in iPSCs induction and committed differentiation,and how lncRNAs function in cell reprogramming and committed differentiation,remains to be further confirmedTo map pluripotency-associated lncRNAs,we combines multiple sequencing results and find a new pluripotent exit-associated lncRNA which combined with pluripotent Oct4 gene promoter,and name it pluripotent exit-associated lncRNA 1,Pelnl for short.Pelnl is differentially expressed in reprogramming,and the knockout of Pelnl can improve the reprogramming efficiency.If Pelnl is overexpressed,the pluripotency of iPSC cannot be maintained.It suggests that Pelnl may play an important role in the pluripotent exit of stem cells.This study will further study the structure,cell location and function of Pelnl,and the specific mechanism that Pelnl regulates the pluripotent exit of stem cells and somatic reprogramming,and finally elucidate the regulation mode of cell fate mediated by lncRNA.In order to achieve the ultimate goal of manipulating and controlling the fate of cells,promote the clinical transformation of induced stem cell productsMethods:1?RNA sequencing was performed on the unprogrammed somatic cells and the programmed iPSCs respectively,and these twoRNA-seq databases were compared to screen out the highly expressed lncRNA in the unprogrammed somatic cells.By using the method of chromatin lncRNA in situ reverse-associated trap sequencing(CLIST-Seq),pluripotent exit-associated lncRNAs with pluripotent Oct4 promoter binding were found.These two databases were integrated to select the pluripotent exit-associated lncRNA Pelnl.Then,the target of LncRNA Pelnl binding to DNA in the genome was verified by reverse validation of RNA reverse transcription-associated trap sequencing(RAT-seq)2?RT-PCR was used to detect the expression differences of lncRNA Pelnl in the reprogramming process and in different cells and tissues.The expression changes of lncRNA Pelnl in the differentiation process of embryonic stem cells and its relationship with other pluripotent factors were observed through the embryoid differentiation experiment.The subcellular localization of lncRNA Peln1 was determined by using two methods:RNA FISH and extracellular nuclear separation experiment.cDNA terminal rapid amplification technique was used to obtain the full length information of lncRNA Peln1.3?The function of lncRNA Pelnl was verified by overexpression experiment,knockdown experiment and reprogramming experiment.After the synthesis of lncRNA Pelnl overexpression plasmid and transfected into mouse embryonic stem cells E14,the changes of cell morphology and the effect on the expression of pluripotent gene were observed.lncRNA Pelnl shRNA knockdown plasmid was synthesized and transfected into dox-induced mouse fibroblasts containing OSKM factor to prevent Pelnl expression.Then,induced reprogramming experiment was conducted to observe whether the reprogramming efficiency was improved4?The target of lncRNA Pelnl was verified by RAT technique and RNA-DNA Fluorescence in situ hybridization(RNA-DNA FISH).The effect of lncRNA Peln1 on chromatin spatial structure was verified by chromatin conformational capture(3C).Methylation experiments were conducted to verify the effect of lncRNA Pelnl on the methylation of Oct4 promoter region.Methods such as chromatin immunoprecipitation assay(ChIP),RNA Binding Protein immunoprecipitation(RIP)and RNA-protein in vitro incubation were used to clarify the specific mechanism of lncRNA Pelnl regulating Oct4 expressionResults:1?Combining CLIST-Seq and RNA-seq databases,find out pluripotent exit-associated lncRNA Pelnl:RNA-seq showed that lncRNA Pelnl was differentially expressed in unprogrammed cells and iPSCs,and the expression level was high in unprogrammed cells.CLIST-Seq results showed that lncRNA Pelnl was specifically enriched in the promoter region of pluripotent factor Oct4 RAT-seq showed that the promoter and 3 'enhancer regions of Oct4 were strongly bound to lncRNA Pelnl2?The results of RT-PCR suggest lncRNA Pelnl expression in the process of reprogramming is diversity,in iPSCs and E14,Pelnl almost no expression,and in unprogrammed cells and somatic cells,Pelnl express high;The results of embryoid differentiation experiment showed that the expression of lncRNA Pelnl was up-regulated with the differentiation of stem cells,contrary to the expression trend of pluripotent factors such as Oct4,Sox2 and Nanog.;Subcellular localization suggested that Pelnl was located in the nucleus.The full-length sequence information of lncRNA Pelnl was obtained by RACE technology.The 3 '-end sequence was consistent with that provided by the current website,and the 5'-end sequence was slightly different from the website report3?In the overexpression experiment,when the expression level of LncRNA Pelnl was increased in pluripotent stem cells,the expression levels of key pluripotent genes Oct4,Sox2,Nanog,etc.,all decreased,and E14 was differentiated.The immunofluorescence staining of Oct4 was negative,making it difficult for cells to maintain pluripotency.In the knockdown experiment,knocking down lncRNA Pelnl at the early stage of reprogramming can improve the reprogramming efficiency and the pluripotency of iPSCs.4?RAT experiment and RNA-DNA FISH experiment proved that Oct4 was the target of lncRNA Pelnl,and lncRNA Pelnl could bind to the promoter and 3 enhancer regions of Oct4.3C experiments showed that in E14 cells,Oct4 promoter and 3'enhancer regions could form chromatin inner ring structure in space,which was destroyed after Pelnl was overexpressed in E14 cells.Methylation experiments proved that after Pelnl was overexpressed,methylation level of Oct4 promoter region of E14 cells increased.ChIP experiment proved that lncRNA Pelnl can promote DNMT3a binding to Oct4 promoter and 3'enhancer region.The RIP experiment further confirmed that lncRNA Pelnl can bind to DNMT3a and suggested that the 3 'end of lncRNA Pelnl is more obviously bound to DNMT3a.In vitro co-incubation of RNA-protein also demonstrated that lncRNA Pelnl could bind to DNMT3a at the 3 'terminal of lncRNA Pelnl.Conclusions:1?LncRNA Pelnl is a new lncRNA located on chromosome 6 of mice.It is located in the nucleus and is differentially expressed in the process of somatic cell reprogramming.The expression gradually increases during the process of stem cell pluripotent exit,and is negatively correlated with the expression of pluripotent factors Oct4,Sox2,Nanog,etc.2?LncRNA Pelnl can promote the pluripotent exit of stem cells.After overexpression of lncRNA Pelnl,E14 can differentiate and it is difficult to maintain pluripotency,and the expression of key pluripotency gene is down-regulated accordingly.LncRNA Pelnl can also hinder reprogramming.Knocking down lncRNA Pelnl at the early stage of reprogramming can improve reprogramming efficiency and improve the pluripotency of iPSCs.3?Oct4 is the target of lncRNA Pelnl.LncRNA Pelnl regulates Oct4 expression by recruiting DNMT3a methylation enzyme,destroying the chromatin inner ring structure between Oct4 promoter and 3 'enhancer,and promoting DNA methylation in Oct4 promoter regionThe innovation points:1.The research object is new:LncRNA in reprogramming of the research is still in its infancy,this study found that LncRNA Pelnl is a new IncRNA located on chromosome 6 of mice,we named it pluripotency exit associated IncRNA1,referred to as "Peln1",it binds to the promoter and enhancer of the pluripotent related gene Oct4,and is differentially expressed during reprogramming,this study first confirmed Pelnl can promote iPSC Pluripotency,affect the efficiency of reprogramming2.Research on "mechanism" is new:This research take "the IncRNA-intrachromoosomal loop-pluripotent genes regulation-pluripotent exit" as the main line,it was first discovered that IncRNA Pelnl could regulate Oct4 expression by recruiting DNA methylase,affecting the intrachromoosomal loop of pluripotent factor Oct4,it provides a new way to improve the efficiency of somatic cell reprogramming and control the differentiation of stem cells.