The Functional Study Of P27^kip1 Gene In Erythropoiesis

PurposeRed blood cells are an important component of blood.Its main function is to transport oxygen through blood circulation to various parts of the body to maintain good health of the body.However,the lifespan of adult mature red blood cells is around 120 days.In order to maintain the proper number of red blood cells in the body,about 2 million red blood cells are produced in adult bone marrow every second.This process is known as erythropoiesis.The erythropoiesis begins with the hematopoietic stem cells,and goes through three major stages to form the fully functional mature red blood cells:the early differentiation of the erythroid,the final differentiation of the erythroid and the enucleation.This is a complex process that is tightly regulated by various mechanisms.Any dysregulation of the erythropoiesis process may cause different types of blood disorders.By going through patients with various blood diseases,we found that p27^kip1?hereinafter abbreviated as“p27”?is commonly mutated in many blood diseases such as polycythemia vera disease.p27mutation often leads to reduced expression of the mRNA.Many studies have reported the association between p27 and erythroid-related hematopathy.Therefore,it is very important to study p27 gene function in erythropoiesis.MethodsFirst,the bioinformatics method was used to analyze the gene structure,protein structure,and the expressions of p27 and its family.p27 knock-down vector was successfully constructed and RT-qPCR was used to verify the knock-down efficiency.Blood cell count,benzidine staining and flow cytometry were used to examine the effects of p27 knockdown on proliferation,differentiation,apoptosis,cell cycle and enucleation of K562,HEL and CD34⁺cells.ResultsFirst,bioinformatics analysis show that the phylogenetic tree of p27 gene and its family members have 6 big branches.Differences in evolutionary distance between members of different branches are smaller,and members of one branch are almost the same species.The amino acid sequence alignment shares 40%-60%similarity.It shows that the conservation of sequences in animals is significantly higher than that of plants.There are several functional domains with higher conservatism in structure.In addition,peripheral blood RNA-seq data showed that p27 gene expression was low in the early differentiation stage,but it was significantly increased in terminal differentiation.In the functional analysis,p27 knockdown K562 and HEL cells showed accelerated cell growth but slow cell differentiation comparing with the control group from day 1 to day 4.Apoptosis was no change.Furthermore,we sorted CD34⁺cells by magnetic beads separation from peripheral blood and cultured for continuous 21 days.Knockdown of p27 gene also slightly accelerated cell growth.There was no significant change of erythroid early differentiation in the BFU-E?CD34⁺CD36^-?and CFU-E?CD34^-CD36⁺?from day 6 to day 7.The expressions of Band 3 and?4 integrin were reduced in the experimental group from day 9 to day 15of terminal differentiation.In addition,the positive cells of Syto16 stain also gradually decreased from day 14 to day 20.We also found abnormal nuclei appeared in the MGG stain from day 14 to day 16.ConclusionFirstly,p27 and its family members are widely found in plants and animals,and are more conserved in animals.Its functional domains are also highly conserved in evolution.RNA-seq data showed that the p27 gene was highly expressesed in the terminal differentiation stage.Second,in K562 and HEL cell lines,knockdown of p27promotes cell growth,inhibits cell differentiation,promotes cell cycle progression but does not change apoptosis.Finally,in CD34⁺cells,p27 knockdown only mildly promotes cell growth,and has no effect on the erythroid early differentiation process.p27 knockdown inhibits the terminal differentiation process as well as inhibits the enucleation.p27 knockdown cells have abnormal nuclei.