| Purpose:Red blood cells(RBCs)are the most abundant cells in the human body and play critical roles in oxygen and carbon dioxide transport.Erythropoiesis is a multi-stage process that can be subdivided into 3 stages:early erythropoiesis,terminal erythroid differentiation,and reticulocyte maturation.This process is accompanied by decreases in cell size,increase in hemoglobinization,membrane reorganization,loss of intracellular organelles and enucleation.Lysosomes is the single-membrane organelles which contain numerous acid hydrolases.Lysosome are now known to play central roles in macromolecule degradation,secretion,plasma membrane repairment,cell signal transduction,cell homeostasis,energy metabolism and many other fundamental cellular processes.The lysosome contains more than 60 soluble acid hydrolases,and the acidification of lysosome is the most important biological characteristic.TPP1 is a serine carboxyl lysosomal protease.TPP1 is a unique enzyme with both tripeptidyl peptidase and endopeptidase activities for certain substrate specificity,and it is the only hydrolase with tripeptidyl peptidase activity identified to date in the lysosomes of mammalian cells.Analysis of transcriptome profile of human erythroid showed that the expression of TPP1 dramatically upregulated during the terminal stage of human erythropoiesis,suggesting TPP1might plays important roles in the regulation of human termimal erythropoiesis.In the present study,we explored sh RNA-mediated knockdown approach in human CD34~+cells to study the role of TPP1 in human erythropoiesis.By combing using the Multi-omics strategy and gene fuction study method,we studied the effects and mechanism of TPP1 deficiency on cells proliferation differentiation,apoptosis and enucleation during normal human erythropoiesis.By further elucidating the roles of TPP1,our current study will not only provide help for the constructin of regulatory net work of human terminal erythropoisis,but also will provide important data support for the understanding of human erythropoisis.Methods:1.Knockdown TPP1 expression during human erythropoiesis.We constructed sh RNA knockdown vectors.Then we performed lentiviral mediated transfection to human cord blood derived from CD34~+cells and K562 cells.Knockdown efficiency of TPP1 was detected by q RT-PCR and Western blotting.2.Effects of TPP1 knockdown on cell proliferation,differentiation,apoptosis,and enucleation during erythropoiesis.During erythropoiesis,we checked cell proliferation by counting cell number.The differentiation of BFU-E and CFU-E was determined by checking the surface expression of GPA,IL-3R,CD34 and CD36 using flow cytometry.During terminal erythroid differentiation,the differentiation was assessed by GPA,?4-integrin and Band 3 surface marker using flow cytometry.The apoptosis was measured by Annexin V using flow cytometry.The enucleation of erythroblasts was examined by Hoechst 33342 using flow cytometry.3.Analysis of transcriptome sequencing and proteome sequencing to explore the relevant mechanisms after TPP1 knockdown.We performed the transcriptome sequencing to analyze the differentially expressed genes and signal pathways between the normal and TPP1 knockdown erythroid cells.Meawhile,we used proteome sequencing to analysis the differentially expressed proteins.By combining analysis of the sequencing results,we tried to determine the key substrates that were accumulated in TPP1 knockdown cells,further established the regulation pathway of TPP1 during erythropoiesis.4.Impact of TPP1 deficiency on the status of lysosome and autophagy.To observe the effect of TPP1 deficiency on the lysosome and autophagy status,we stained the normal cells and the TPP1 deficent cells with antibody against erythroid cell surface marker GPA,agianst lysosome marker LAMP1/autophagy marker LC3?together with the nuclear dye Hoechst 33342,and took pictures using immunofluorescence microscope.The expression of those markers also were determined using flow cytometry and Western Blotting.5.The effect of TPP1 deficienby on p53 signal pathway.The analysis of the results of RNA-Seq and Proteomics data showed that knock-down of TPP1 induced dramatically upregulation of p53 and some key down-stream transductors.Therefore,in order to further elucidate the mechanism underlying the TPP1 deficiency induced cell apoptosis,we performed Western Blot to check the expression and activation of the key molecules of p53 signal pathway.6.The effect of TPP1 knockdown on insulin receptor.The analysis of the results of RNA-Seq and Proteomics data showed significant change of the surface expression of insulin receptors,which were also confirmed by q RT-PCR and Western Blotting.In order to further elucidate the mechanism underlying the impairment of the critical cell events,such as cell differentiation,cell apoptosis and enucleation,that were induced by TPP1 deficiency,we performed flow cytometry and Western Blot to check the status of insulin receptors.Results:1.Knockdown TPP1 during human erythropoiesis in vitro.Human CD34~+hematopoietic stem cells were isolated from umbilical cord blood.K562 cells were cultured according the protocols of our lab.TPP1 was knockdown using lentivirus mediated sh RNA strategy.Knockdown efficiency of TPP1 was above50%during the whole process of human erythropoiesis.2.TPP1 knockdown inhibited cell proliferation,increased apoptosis and impaired enucleation.In order to explore the role of TPP1 during eythropoiesis,we checked the cell proliferation,cell differentiation,cell apoptosis and enucleation of the TPP1 deficient erythroid cells.The results showed that TPP1 knockdown severly impaired the cell proliferation,cell apoptosis and enucleation,but showed no significant influence on the proliferation of erythroid progenitors and erythroid terminal differentiation.However,compared with the control group,TPP1 knockdown K562 cells showed no significant changes of cell.These results indicated that TPP1 plays important roles in the regulation of process of human erythropoiesis.3.Transcriptome sequencing and proteomic analysis showed the differences between TPP1 knockdown cells and normal cells.In order to investigate the mechanism underlying the cell events disorder that were cuased by TPP1 knockdown,we performed transcriptome sequencing and proteomic analysis of TPP1 knockdown cells and normal cells,respectively.GO analysis of the transcriptome sequencing results showed that TPP1 knockdown mainly affect the homeostasis and metabolism.KEGG enrichment analysis showed that there were sinificant difference in some certain biological process,such as autophagy,lysosome,p53 signaling pathway and insulin resistance.4.TPP1 knockdown led to increased lysosome numbers and do no effect autophagy.Since TPP1 is a lysosomal hydrolase,we therefore checked the influence of TPP1 deficiency on the lysosome status during erythropoiesis.Flow cytometry and Western Blotting analysis showed that the lysosome number was significantly upregulated in TPP1 deficient erythroid cells that of the normal cells.The results of immunofluorescence staining showed that there was no significant change of the lysosomes morphology,but the relative fluorescence intensity of lysosome marker was increased.Take together,these results indicated that knockdown of TPP1 cause the increasing of the lysosomes number,which might be the compensation for the defects of degradation capability.Since it has been well established that lysosome play critical roles in the regulation of autophagy that have been proven to be critical for the late stage erythroid development process.To determine the effect of TPP1 eficiency on cell autophagy,we checked autophagy status using immunofluorescence staining and flow cytometry.The results showed that there were no significant differences of the autophagy process between TPP1 knockdown and normal cells.However,since the autophagy is a dynamic process,further experiment should be performed to chase the“autophagic flux”in order to clarify the roles of TPP1 in the regulation of autophagy.5.p53 inhibitor could not rescue the defects of cell proliferation and increased apoptosis.In order to further explore the mechanism of cellular abnormality caused by TPP1 knockdown,we analyzed transcriptome sequencing and proteome sequencing results and found that the expression of p53 was significantly decreased.This result was also confirmed by Western Blot assay.p53 inhibitor was applied to determine whether cell proliferation and apoptosis can be rescued after TPP1 knockdown.The results showed that p53 inhibitor could not rescue the impaired cell proliferation and increased cell apoptosis,indicating that the increased cell apoptosis caused by TPP1knockdown was not through the transduction of p53 signaling pathway.Further research should be performed to investigate the detailed mechanisms.6.TPP1 knockdown resulted in a significant decrease in insulin receptor expression.Analysis of RNA-seq results showed a significant change of insulin-related signaling pathways.Meanwhile,the proteomic analysis showed that the expression of insulin receptor level was reduced after TPP1 knockdown.We detected insulin receptor expression using q RT-PCR and Western Blotting.The expression of insulin receptor was found to be decreased in both m RNA and protein levels which were consistent with multi-omic analysis.Since the signaling pathway that was activated by insulin receptor played critical roles in the regulation of human erythropoiesis by modulating cell metabolism,cell growth and cell differentiation,we proposed that the deceased expression of insulin receptor cuased by TPP1 deficiency might be the important mechanism for the cell function impairment during the terminal stage of human erythropoiesis.Conclusion:In conclusion,TPP1 knockdown severly impaired the cell proliferation,cell apoptosis and enucleation during human erythropoiesis.Although the expression of p53 was increased after TPP1 knockdown,the apoptosis is not directly caused through p53 signaling pathway.The deficiency of TPP1 leads to an increase of lysosomes number,which may be related to the decline in the degradation ability.TPP1 knockdown may cause changes in insulin receptor-related pathways,but the detailed mechanism needs to be further elucidated. |
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