The Role Of Long Non-coding RNA Oslr9 In The Regulation Of Stem Cell Pluripotency And Its Mechanism

Background:Terminally differentiated somatic cells can be reprogrammed into stem cell-like pluripotent state by nuclear transfer,cell fusion,transduction of pluripotency factors,etc.,enabling them to self-renew indefinitely and differentiate into all types of cells in the body.This process is called somatic cell reprogramming(SCR).The emergence of this technology has provided great prospects for regenerative medicine,individualized treatment of diseases and drug screening.However,no matter which method is used,somatic reprogramming is time-consuming and low efficiency,thus limiting its application potential in medicine and other areas.At present,the mechanisms of pluripotent stem cell self-renewal,pluripotency maintenance and reprogramming process is not clear,but it is crucial to maintain stem cell pluripotency and improve the efficiency of reprogramming.Studies have shown that the maintenance of pluripotency or differentiation into any germ layer depends on the regulation of thousands of gene expression through epigenetics,transcription,and post-transcriptional regulation.Long noncoding RNA(lnc RNA)is a class of RNA that does not have the ability to encode proteins but is involved in many biological processes.Now accumulate evidence indicate that lnc RNAs participate in the regulation of gene expression by gene imprinting,chromatin remodeling and so on,and was found to be involved in the regulation of stem cell pluripotency maintenance and cell differentiation.At present,many experts and scholars are devoted to studying the epigenetic modification of lnc RNA in stem cell pluripotency,so as to find a way to improve the efficiency of reprogramming.In our previous study,we used a modified chromatin-RNA in situ reverse transcription sequencing technique to capture lnc RNAs that bind to the pluripotency genes Oct4 and Sox2 promoters.By combining the results of analysis of CRIST-seq and RNA-seq,a long non-coding RNA that binds to the Oct4,Sox2 promoter was discovered and named Oslr9.We speculate that Oslr9 is closely related to stem cell pluripotency and has carried out various studies on it.Objective:This study aimed to clarify basic information such as the differential expression,full-length sequence and subcellular localization of lnc RNA Oslr9,and to explore the role of lnc RNA Oslr9 in the regulation of stem cell pluripotency and its epigenetic regulatory mechanism.Methods:1.The combined use of chromatin-RNA in situ reverse transcription sequencing(CRIST-seq)and RNA sequencing(RNA-seq)revealed stem cell pluripotency associated long non-coding RNA Oslr9.2.Rapid amplification of c DNA ends(RACE)technology was used to determine the full length and the sequence of Oslr9.3.Real time quantitative polymerase chain reaction(RT-q PCR)was used to determine the expression levels of Oslr9 and three stem cell pluripotent core factors(Oct4,Sox2,Nanog)during cell reprogramming and embryonic stem cell differentiation.4.The location of Oslr9 in cells was determined by RNA fluorescence in situ hybridization and nuclear and cytoplasm fractionation assay.5.Knockdown Oslr9 in mouse induced pluripotent stem cells(mi PSCs)and examine its effect on cell function: construct two plasmids containing short hairpin RNA structures and stably transfect into mi PSCs,using RT-q PCR to detect the expression of Oslr9,Oct4,Sox2 and Nanog in the mi PSC group,mi PSC+p Green vector group and mi PSC+sh Oslr9 group.The cell proliferation ability of mi PSC group,mi PSC+p Green vector group and mi PSC+sh Oslr9 group were determined by MTT assay;the pluripotency state of cells after Oslr9 down-regulation was assessed by cell immunofluorescence staining.6.Oslr9 activates the stem cell pluripotency factors: Oslr9 overexpression plasmid was constructed and stably transfected into mouse fibroblasts,and detected the expression of Oslr9 and Oct4,Sox2,and Nanog in m FIB,m FIB+Ds Red vector group and m FIB+Oslr9 group by RT-q PCR;The dual-luciferase reporter assay system was used to determine the role of Oslr9 in activating the promoter of stem cell pluripotency factors.7.Reveal the binding of Oslr9 to other DNAs by RNA reverse transcription-associated trap(RAT).8.The Bisulfite sequencing PCR was used to determine the methylation status of the Oct4 promoter and enhancer elements.9.The interaction between Tet protein and Oct4 gene regulatory elements was detected by chromatin immunoprecipitation(Ch IP)technology.10.The binding of Oslr9 to Tet protein was confirmed by RNA immunoprecipitation(RIP)technology.11.Chromatin conformation capture(3C)was used to determine the three-dimensional regulation of Oslr9 on the Oct4 gene enhancer-promoter looping structure.Results:1.Identification of Oslr9 as a pluripotency associated lnc RNA by the analysis of CRIST-seq and RNA-seq.RNA-seq indicated that Oslr9 was highly expressed in mouse induced pluripotent stem cells(mi PSCs)but silenced in mouse fibroblasts;CRIST-seq showed that Oslr9 bound to pluripotency gene Oct4 and Sox2 promoter.3'-and 5'-RACE showed that there were two transcription variants of Oslr9,one of which had the same sequence as Gm28268,and the other had two exons and the 5 'end was different from Gm28268 and was 914 bp in length.2.Oslr9 was upregulated in mouse induced pluripotent stem cells(mi PSCs)and mouse embryonic stem cells(m E14)but silenced in mouse fibroblasts(m FIBs)and cells that have not been successfully reprogrammed to i PSC(m URCs);During the process of embryonic body formation,the expression level of Oslr9 is downregulated,in parallel with Oct4,Sox2 and Nanog.3.Nuclear and cytoplasm fractionation assay and RNA-FISH showed that Oslr9 mainly located in the nucleus.4.After the knockdown of Oslr9 in mi PSC,the expression of pluripotency genes Oct4,Sox2 and Nanog was down regulated and the proliferative ability was decreased compared with the p Green vector and mi PSC group.Morphology indicated enlarged mi PSCs after down-regulating Oslr9 expression,the cells showed a flat and elongated morphology,and immunofluorescence staining showed that the expression of the pluripotency factor Oct4 and Nanog was decreased.5.After the overexpression of Oslr9 in mouse fibroblasts,the expression of pluripotency genes Oct4,Sox2 and Nanog was upregulated;dual luciferase reporter assay showed that the promoters of Oct4,Sox2 and Nanog in cells that transfected with Oslr9 overexpression plasmid are apparently activated compared to control group.6.The methylation test results showed that significant demethylation occurred at the Oct4 gene promoter and 5'-proximal enhancer in fibroblasts transfected with Oslr9 overexpression plasmid compared to the control group;chromatin immunoprecipitation suggested Oslr9 can mediate the binding of Tet1 protein to Oct4 gene regulatory elements;RNA immunoprecipitation and sequencing results show that Oslr9 binds to Tet1 via exon 1.7.CRIST-seq result showed that the first exon of Oslr9 binds to the Oct4 promoter,and multiple exons of Oslr9 bind to the Sox2 promoter.RNA reverse transcription-associated trap experiment confirmed Oslr9 bind to the promoter,enhancer and exon of the Oct4 gene.Chromatin conformation capture(3C)experiments indicated that Oslr9 is involved in the formation of multiple loop structures of the promoter and enhancer of Oct4 gene,shortening the distance between promoter and enhancer,thus enhancing the expression of pluripotency gene Oct4.Conclusions:1.Oslr9 is a long intergenic noncoding RNA located in the X chromosome that binds to the Oct4-Sox2 gene promoter,it has two transcription variants.2.Oslr9 mainly plays its role in the nucleus,and is highly expressed in mi PSC and m ESC,but hardly expressed in m FIB and m URC.Its expression level is positively related to the pluripotency genes.3.Oslr9 is closely related to stem cell pluripotency and reprogramming,it has the function of maintaining stem cell pluripotency and promoting the expression of pluripotency genes.4.Oslr9 recruits the Tet1 protein through the first exon,induces demethylation of the Oct4 promoter and 5'-proximal enhancer,and activates the promoter and 5'-enhancer of Oct4,thereby promoting the expression of the pluripotency gene Oct4.5.Oslr9 can help with the formation of 5'enhancer-promoter and 3'enhancer-promoter intrachromosomal looping structure which located in Oct4 gene,thus making the distance between enhancer and promoter get closer and promoting the expression of Oct4 gene.