Splicing Factors SRSF2 And SRSF1 Regulate The Differentiation Of Human Embryonic Stem Cells Into Endothelial Progenitor Cells

Research background:Alternative splicing,as a key factor to increase the complexity of the gene transcriptome and protein diversity,is essential for regulating individual growth and development.More and more studies have shown that the components involved in alternative splicing play an important role in regulating hematopoiesis.Alternative splicing disorders and splicing factor mutations can cause hematopoietic system disorders and eventually hematologic diseases.However,the regulatory effect of alternative splicing on early hematopoietic development remains unclear.The preliminary research of the subject found that there are extensive and dynamic alternative splicing events in the process of monolayer hematopoietic differentiation,and the splicing factor switch exists in the process of differentiation from APLNR+mesoderm cells into endothelial progenitor cells.Inhibiting alternative splicing changes the expression pattern of splicing factors.And it specifically affects the generation of EPC(endothelial progenitor cell).During the switch process of the splicing factor,it was found that the expression of the splicing factor SRSF2,which is a member of the SR protein family,changed significantly.There have been many reports of SRSF2 for regulating blood system diseases,but its function in early hematopoietic development is still unclear.SRSF1 is also a classic member of the SR protein family,and its expression pattern is similar to that of SRSF2 in the process of hematopoietic differentiation.The function of regulating hematopoietic differentiation also needs further research.Research objective:Explore the function and regulation mechanism of splicing factors SRSF2 and SRSF1 in the early hematopoietic development of hESC(human embryonic stem cell).Research methods:In order to explore the functions of splicing factors SRSF2 and SRSF1 in the early hematopoietic development of human embryonic stem cells,we first established inducible overexpression stable cell lines,and specifically overexpressed SRSF2 and SRSF1 at the stage of APLNR+mesoderm cell differentiation to EPC.Flow cytometry was used to detect CD31+CD34+endothelial progenitor cells to explore the effect of overexpression of SRSF2 and SRSF1 on hematopoietic differentiation.Then,by establishing stable cell lines with inducible knockdown of SRSF2,DOX was added periodically to induce knockdown during hematopoietic differentiation,and CD31+CD34+endothelial progenitor cells were detected by flow cytometry on day 5 to explore the effect of staged knockdown of SRSF2 on EPC.In addition,RNA interference technology was used to add siRNA to knockdown SRSF1 in the EPC generation stage,and flow cytometry to detect the generation of EPC.Finally,the alternative splicing reporter system was used to verify the molecular mechanism of splicing factors SRSF2 and SRSF1 regulating EPC production.Research results:1.Successfully constructed stable cell lines that induced overexpression of SRSF2 and SRSF1.The overexpression of SRSF2 in APLNR+mesoderm cells inhibits the generation of EPC,while the overexpression of SRSF1 promotes the production of EPC.2.Successfully constructed stable cell lines with inducible knockdown of SRSF2.The knockdown of SRSF2 in APLNR+mesoderm cells promotes the generation of EPC.3.Knock down SRSF1 to inhibit the production of EPC at the stage of differentiation of APLNR+mesoderm cells into EPC by RNA interference technology.4.In terms of molecular mechanism,SRSF2 regulates EPC by promoting the production of the long isoform of NUMB;and SRSF1 regulates EPC by promoting the production of the short isoform of NUMB.Research conclusion:Splicing factors SRSF2 and SRSF1 regulate the production of EPC during the early hematopoietic development of human embryonic stem cells by regulating the alternative splicing of NUMB.