Function Of Long Non-coding RNA In Hematopoietic Stem Cell Development

Hematopoietic stem cell(HSC)is generally considered as the cornerstone of hematopoietic system.It has the function of continuously producing all mature blood cells in the whole life cycle of organisms,and can completely rebuild adult hematopoietic system and immune system.It is one of the adult stem cells with the longest and deepest research history.HSC transplantation is commonly used in clinical to treat various blood system diseases,hereditary diseases,some solid tumors and autoimmune diseases.Moreover,this treatment has good therapeutic effects.However,due to the limited source of HSC and difficulties of amplification in vitro,many patients have lost the opportunity to receive treatment.In order to solve these problems,scientists have attempted to induce pluripotent stem cells to differentiate into HSC directionally with small molecule compound.But being lack in systematic scientific understanding of the basic regulatory mechanisms of HSC development makes induction of HSC regeneration in vitro still unsuccessful.Therefore,it is of great important to explore the occurrence and development of HSC and its regulatory mechanism.At the early stage of mouse embryonic development,the first HSC capable of long-term repopulation of adult hematopoietic system is produced on the embryonic day 10.5.It originates from a number of vascular sites including yolk sac,head and aorta-gonad-mesonephros(AGM),etc.In recent years,studies have found that the generation of HSC in AGM region,one of the most important blood-producing sites,undergoes a process of endothelial-hematopoietic transition(EHT)from blood-producing endothelial cells,then goes through two types of pre-hematopoietic stem cell,namely type ? pre-HSC and type ? pre-HSC,eventually further develops and matures into HSC.The time of HSC development is short,the number of key intermediate cell populations such as pre-HSC and HSC is extremely rare,and there is a lack of specific surface marker molecules to enrich these cell populations,which makes it extremely difficult to study the origin and molecular regulation mechanism of HSC.Up to now,the known regulatory mechanism of HSC development only focus on the regulation of signal pathways and transcription factors,for example,transcription factors Runx1(and its downstream transcription factors Gfi1 and Spi1),Gata2,etc.,which are indispensable for EHT.Transcription factor Hoxa3 maintains endothelial characteristics of endothelial cells and stops endothelial cells from transiting to hematopoietic cells by inhibiting the expression of important hematopoietic transcription factors and promoting the expression of endothelial transcription factors.Transcription synergetic molecule Smad4 has also been proved to negatively regulate this process.In addition,signaling pathways such as BMP/TGF-?,Notch,Wnt,TNF-? and Hedgehog play an essential role in EHT.Compared with the relatively thoroughly studied coding genes and signal pathways,the dynamic expression profile and physiological function of long non-coding RNA(lncRNA)during HSC development have not been reported so far.Previous research had established an incubation-induced transplantation experimental system with single cell for the first time in the world,and eventually found specific surface marker CD201 through large-scale screening of various surface marker combinations,and achieved accurate capture of pre-HSC up to 30%.At the same time,combined with single-cell transcriptome sequencing technology,the dynamic molecular expression of related cell population in the whole life cycle of HSC are systematically revealed,thus realizing accurate research at the single-cell level.Based on the above high-throughput sequencing data of single-cell transcriptome during HSC development,this study further identified 7,312 lncRNA in the six cell populations through systematic bioinformatics analysis,and mapped the dynamic expression pattern of single-cell lncRNA molecules during HSC development for the first time.In addition,the temporal and spatial expression characteristics of lncRNA in six continuously developing cell populations,correlation with neighboring protein coding genes,and stage-specific genes are systematically disclosed.And physiological functions of relevant lncRNA are predicted by co-locating or co-expressing with loci of important protein coding genes.Pearson correlation analysis shows that lncRNA has the strongest correlation with its first neighboring protein coding gene.Principal Components Analysis(PCA)shows that lncRNA is easier than mRNA to distinguish different spatiotemporal similar cell populations,revealing that lncRNA may have stronger stage specificity during hematopoietic stem cell development,and further uncovering that lncRNA plays an important role in the whole process.At the same time,PCR and Sanger sequencing were carried out on 401 completely new and unannotated lncRNA analyzed in the study,and a series of completely new and unannotated lncRNA molecules were identified during HSC development,confirming the potentially important function of lncRNA in regulating HSC development.Subsequently,10 potential functional candidate lncRNAs were screened according to three strict bioinformatics screening criteria,and 6 lncRNA that may play a role in embryonic hematopoietic development were found through colony forming units in culture(CFU-C)in vitro.These 6 lncRNA are H19,AI66270,4933439C10 Rik,Gm15135,Gm17275 and 1700113A16 Rik respectively.Furthermore,the research strategy of conditional gene knockout and AGM direct transplantation experiment emphasizes that lncRNA-H19 is crucial to the occurrence of HSC in AGM region.Sequencing of single cell transcriptome shows that the expression of important hematopoietic transcription factors such as Runx1 and Spi1 are significantly down-regulated after lncRNA-H19 deletion.Pseudo-timing analysis also proves that lncRNA-H19 deletion blocks the EHT.Analysis of genome-wide methylated high-throughput sequencing data shows that the deletion of lncRNA-H19 makes the promoter region of important hematopoietic transcription factors(including Runx1 and Spi1,etc.)hypermethylated,and the promoter region of endothelial cell-specific expression transcription factors such as Sox17 hypomethylated,suggesting that lncRNA-H19 may down-regulate or up-regulate expression by regulating the methylation levels of important hematopoietic transcription factors and endothelial cell-specific transcription factors,thus blocking the EHT.Because the location of lncRNA determines their own functions,mechanism studies have found that the cytoplasmic localization of lncRNA-H19 regulates the methylation level of the target gene by inhibiting the activity of the methylation regulatory molecule S-adenylhomocysteine hydrolase(SAHH).Interestingly,we also find that lncRNA-H19 is independent of the previously reported product mir-675 produced by lncRNA-H19 as a microRNA precursor(pri-miR)but by its own action.Previous literature reported that mir-675 maintained the quiescence state of adult HSC by regulating Igf1r-Igf2 signaling pathway.To sum up,our study reveals lncRNA which plays an important role in HSC development for the first time in the world,and systematically expounds the completely different functions and regulation modes of lncRNA-H19 in embryonic HSC development and adult HSC homeostasis maintenance.These understandings will provide important enlightenment for a comprehensive understanding of the mechanism of lncRNA regulating important biological processes,and the single-cell lncRNA map of HSC development will also provide important databases and references for the study of HSC development and regeneration mechanism.