Constructing Globin Gene Co-expression Network Based On Single-cell Transcriptomic Profile Of Human Terminal Erythroblast

Background:Human terminal differential erythroblast is a key stage of erythrocyte development and maturation,and a detailed study of gene transcription activity at this stage helps to understand the dynamic changes of the erythrocyte maturation process.The rapid development of transcriptome research technologies,such as single-cell RNA sequencing technology,has made great progress in recent years,allowing researchers to deeply describe and analyze the cell composition and gene signature expression of heterogeneous tissues,so as to have the opportunity explore regulatory relationships between genes.However,there is currently no single-cell sequencing of terminal-stage bone marrow derived in vivo to elucidate the process of red blood cell maturation and differentiation from the single-cell level.Objectives:In this study,we used three human health adults' bone marrow CD235a+ cells as basic materials on which single-cell RNA-sequencing(scRNA-seq)was conducted to explore the cell composition and gene expression of the terminal-stage of erythroid differentiation.Surrounding the globin genes module at the single-cell level on which a local gene co-expression network was built to discover potential erythroid regulatory factors.Results:We obtained a total of 8,668 effective cells across about 15,000 transcripts by single-cell sequencing.The identification results of terminal differential erythroblasts and pseudo-time analysis showed that the process is continuous.The number of cells in each stage is:85 proE cells,446 basoE cells,2,185 polyE cells,5,952 orthoE cells,in which polyE cells were further divided into transit-polyE,early-polyE and late-polyE while orthoE cells were into early-orthoE and late-orthoE.We found that as differentiation progressed,the number of genes detected decreased while the globin genes proportion increased.Through the globin genes co-expression network,we identified 83 genes and 38 transcription factors which have potential regulatory relationship with globin genes.Conclusion:In this study,CD235a+ cells from bone marrow in vivo were applied single-cell RNA-sequencing technology.We concluded that,(1)traditional identities of terminal-stage cells were assigned,and supplemented by subdividing the polyE and orthoE cells.The identification and subdivision of cell types can provide a clear understanding of the terminal-stage development and a basis for studying the mechanism of different proportions of cells in each differentiation stage contained in the blood of patients with erythrocyte-related diseases.(2)specifically expressed genes in different stages of terminal erythroid differentiation were obtained.These genes will provide gene targets for the molecular mechanism of studying the open expression of globin genes in erythrocytes,nuclear shrinkage and denucleation,and then study the pathogenesis of some erythrocyte-related diseases.(3)based on the pseudo-temporal information estimated from the single-cell gene expression profile,a co-expression network was constructed around the globin genes.Thus,more genes related to the globin expression process were uncovered for further understanding and mining the process of erythrocyte maturation and differentiation,which provides a lot of regulatory details and new clues for identifying and searching for possible genes related to erythrocyte maturation and differentiation.