The Functional Study Of Autophagy Gene ATG13 In Erythropoiesis

Research purposes:Erythropoiesis is a complex and delicate process.There are three stages oferythropoiesis:early erythropoiesis,terminal erythroid differentiation and reticulocyte maturation.Autophagy is a highly conserved lysosomal degradation process,which plays an important role in the clearance of organelles such as ribosomes and mitochondria during the late stage of erythroid terminal differentiation.The autophagy initiation complex consists of ATG13?ULK1?FIP200?and ATG101.ATG13 acts to link ULK1 and FIP200 and increase ULK1 kinase activity.It has been found that ULK1 plays an important role in clearance mitochondria and ribosomes during reticulocyte maturation;FIP200 plays an important role in the maintenance of cellular autonomy of fetal hematopoietic stem cells.ATG13 and ATG101 have not been studied in the erythroid development.It is speculated that ATG13?ULK1?FIP200 and ATG101,which constitute the autophagy initiation complex,play different roles in erythroid and have functional differences.Therefore,the study uses shRNA to study the functional differences of autophagy initiation complexes in erythroid development.Knockdown ATG13?ULK1?FIP200 and ATG101 in K562 cells and knockdown efficiency was detected at the mRNA level.K562 cells were induced to differentiate by hemin.The changes of cell proliferation,apoptosis and differentiation were detected.Then,the ATG13 gene was knocked down in cord blood-derived CD34~+cells to detect cell proliferation,differentiation,apoptosis,and denucleation.By knocking down ATG13?ULK1?FIP200 and ATG101,we studied the different effects on the erythroid development process,and then studied the functional differences of the autophagy initiation complex and the unique function of the ATG13gene.Methods:In order to study the function of ATG13?ULK1?FIP200 and ATG101 in human erythroid development.We searched for the expression of four genes in K562 and the expression of four genes in each phase of RNA-seq results from cord blood-derived CD34~+cells.Secondly,obtain K562 cells that knockdown by ATG13?ULK1?FIP200and ATG101,respectively.Then,the knocked down K562 were induced to differentiate into erythroid cells by hemin.In the four days after induction the growth curve was measured on the cell counts,the cell differentiation was detected by benzidine staining,and the apoptosis was detected by Annexin/7AAD staining.The PRRL-EGFP-LC3B plasmid was constructed and transfected into K562 cells with ATG13 knockdown,at the same time add the autophagy inhibitor BafA1.The green fluorescence spot aggregation was detected by confocal microscopy to detect autophagy levels.CD34~+cells that were isolated from human cord blood and transduced with lentivirus packaged with ATG13 shRNA to knockdown ATG13,and then were cultured in vitro to differentiate into erythroblasts.The knockdown efficiency of ATG13 was detected by quantitative real-time PCR and western blot,the erythroid differentiation process,apoptosis and the efficiency of enucleation were detected with flow cytometery.The cells were counted with a hemocytometer and drawn the growth curve.Cell morphology was observed by MGG staining.The changes of apoptotic genes in CD34~+cells after knockdown of ATG13 were detected by quantitative real-time PCR to explore the causes of apoptosis caused by knocking down ATG13.Results:In K562 cells the ATG13?ULK1?FIP200 and ATG101 were expressed and were differences.The results of RNA-seq showed that ATG13?ULK1?FIP200 and ATG101gradually increased with erythroid differentiation,and were highly expressed in the Poly and Ortho phases.ULK1 knockdown in K562 cells has no effect on cell proliferation,differentiation and apoptosis;FIP200 knockdown in K562 cells has no change in cell proliferation and differentiation,and apoptosis is significant but not obvious on the first day and the second day;ATG101 knockdown in K562 cells has no change in proliferation and apoptosis,differentiation of the experimental group was faster than control group on the first day to the third day,but the differentiation was consistent on the fourth day.ATG13 knockdown in K562 cells,cell proliferation slows,apoptosis increases,and the differentiation is delayed.The phenotypes of knocking down ATG13 in K562 cells were obvious.We further examined changes in autophagy after knockdown of ATG13.K562 cells knockdown with ATG13 and transfecte EGFP-LC3B fusion protein were observed under confocal microscopy.There was no significant difference in green dot aggregation between the control group and the experimental group.However,after adding the autophagy inhibitor BafA1,it can be clearly seen that the autophagy of the ATG13 knockdown group is blocked.It was found that cell proliferation decreased from day 13 and apoptosis also increased from day 13 when knockdown ATG13 in CD34~+cells;cell differentiation was slightly delayed,but not blocked;cell nucleation rate was decreased.The expression of P53?Cytc?Bax?Bak?Smac?Bcl-2?cIAP1?cIAP2?AIF?PARP1?TNF and BNIP3 were detected by quantitative real-time PCR on day 7?day 13?and day 15.It was found that the expression of Bcl-2?cIAP1?cIAP2 and AIF decreased gradually.The expression of P53?Cytc?Bax?Bak and Smac increased gradually.The expression of PARP1?TNF and BNIP3 was unchanged.Conclusion:During erythroid development,ATG13 has different functions from the other three autophagy initiation complex genes ULK1?FIP200 and ATG101.ATG13 can regulate apoptosis by affecting the expression of P53?Cytc?Bax?Bak and Smac.ATG13 may play a special function independent of autophagy in erythroid development.