| With the rapid development of science and technology,nuclear-related technologies are widely applied in production and daily life,while the hazard of people being exposed to ionizing radiation（IR）is also increasing.Bone marrow（BM）is one of the most sensitive and critical target organs for exposure to ionizing radiation in human body.Radiation exposure more than 1 Gy can cause obvious hematopoietic abnormalities in the bone marrow,and larger doses of radiation exposure can even lead to hematopoietic failure.A large number of studies have reported that the homeostatic imbalance of hematopoietic stem cells（HSCs）is the root cause of moderate to severe bone marrow radiation injury,and the accumulation of DNA mutations in HSCs after radiation injury significantly increases the incidence of leukemia,especially myeloid leukemia.Therefore,exploring how to regulate the homeostasis of HSCs has important clinical value for the treatment of moderate to severe radiation injury and leukemia.HSCs are a special class of cells in the blood system with continuous self-renewal and differentiation abilities.HSCs are heterogeneous and can be further divided into long-term HSC（LT-HSC）,short-term HSC（ST-HSC）and multipotent progenitors（MPP）according to their hematopoietic reconstitution ability.Under steady state,the vast majority of LT-HSCs are in a quiescent（G0 phase）cell cycle state,and this characteristic of HSCs is one of the main reasons for maintaining the stability of hematopoietic stem and progenitor cells（HSPCs）pool.Recently,more and more scholars believe that the cell cycle state of HSCs is not only closely related to homeostatic regulation,but also plays vital role in the process of hematopoietic reconstruction after radiation injury in bone marrow.In recent years,numbers of key regulators and signaling pathways have been found to be involved in the homeostatic regulation of HSCs.However,the role and mechanism of cell cycle regulation in this process have not been fully elucidated and further exploration are needed.Cell cycle-dependent kinase 19（CDK19）is one of the key members of the CDK protein family,and is also known as CDK8L/CDC2L6/Cdk11 because of its high sequence similarity with CDK8.In the nucleus,CDK8/19 is involved in regulating cellular stress response,body growth and development,and nervous system function by forming a transcriptional mediator complex（TMC）with MED12,MED13 and cyclin C.Recent studies have found that CDK8/19 expression are significantly increased in breast,prostate and colon cancer,and are closely related to the development and outcome of cancer.In addition,CDK8/19 has been reported to play a crucial role in regulating the proliferation and stress response of human colorectal cancer cells,whether they have similar roles in the hematopoietic system has not been reported.Through the database and pre-experimental results,we observed the relatively high expression of CDK19 in bone marrow HSCs,and this enrichment was more pronounced after ionizing radiation.Consequently,we speculated that CDK19 may be involved in the homeostatic regulation of HSCs and is vital for radiation stress injury in HSCs.In this study,we mainly explored the role and molecular mechanism of CDK19 in regulating HSCs under homeostatic and stress conditions,and verified it with an acute myeloid leukemia（AML）cell line.We first used Quantitative PCR（q PCR）to detect the expression of CDK19 in mouse bone marrow HSPCs under homeostatic and stress conditions.Subsequently,the proportion,cell cycle and apoptosis of bone marrow HSPCs in CDK19-/-mice were detected and analyzed by flow cytometry.More importantly,we demonstrated the effect of CDK19 deletion on the long-term reconstitution ability of hematopoietic stem cells through competitive bone marrow transplantation,reverse transplantation and homing experiments.At the same time,a bone marrow radiation injury model was constructed to observe the effect of CDK19 on HSCs under radiation stress.In addition,we used transcriptome chip,immunoprecipitation（IP）,chromatin immunoprecipitation（Ch IP）and other technologies to further study the underlying molecular mechanism of CDK19 in regulating bone marrow HSC homeostasis and function.Finally,we validated the role and mechanism of CDK19 in two acute myeloid leukemia cell lines,HL-60 and NB-4.The main findings and conclusions are as follows:1.CDK19 is an important factor in maintaining the proliferation ability of HSCs1.1 Database and q PCR results showed that the expression of CDK19 in mouse bone marrow HSCs was significantly increased.CDK8/19 inhibitor（Senexin B）significantly slowed down the expansion of HSCs and inhibit their colony formation after treatment,while CDK19 overexpression can significantly promote the proliferation of HSCs and significantly enhance their long-term hematopoietic reconstitution ability.In conclusion,CDK19 may play an indispensable role in maintaining the proliferation ability of HSCs.1.2 Flow cytometry showed that the proportion and number of HSCs and their subsets in CDK19 knockout mice were significantly reduced.Ki67 and Brd U staining showed that the ability of HSCs to exit quiescence was significantly weakened after CDK19 knockout,while the apoptosis level did not change significantly.These results suggest that CDK19may achieve a homeostasis of HSPC pools by maintaining the proliferative capacity of HSCs.2.CDK19 promotes the recovery of hematopoietic function under stress2.1 In the mouse radiation injury model,CDK19 knockout mice were more sensitive to radiation,and the recovery of peripheral blood mature cells and bone marrow hematopoietic stem cells was significantly slowed down.In addition,the apoptosis level of CDK19-deficient HSCs increased significantly after irradiation;On the other hand,5-fluorouracil（5-FU）treatment significantly reduced the survival of CDK19 knockout mice and significantly delayed the recovery of their peripheral blood mature cell count.The above results indicate that CDK19 has a promoting effect on the recovery of bone marrow hematopoietic function under stress,which may be attributed to CDK19 maintaining the proliferative ability of HSCs.2.2 Experiments such as competitive bone marrow transplantation and post-transfection（overexpression of CDK19）transplantation confirmed that CDK19 is essential for maintaining the long-term hematopoietic reconstitution ability of HSCs.2.3 Homing experiments and reverse bone marrow transplantation show that CDK19maintains the long-term hematopoietic reconstruction ability of bone marrow HSCs through an intrinsic way.3.CDK19 regulates the proliferation and self-renewal ability of HSCs through the p53-p21 signaling pathway3.1 The results of whole gene transcriptome chip showed that after CDK19 knockout,the quiescent-related features in hematopoietic stem cells were significantly enriched,the cell cycle process was significantly inhibited and the p53 signaling pathway was significantly activated.3.2 Further experiments confirmed that the expression level of p53 in CDK19-deficient HSCs did not change obviously,while the expression level of p21increased significantly.After overexpression of CDK19,the expression level of p21 in HSCs was significantly reduced.These results suggest that CDK19 is involved in regulating p21 expression levels in HSCs.3.3 IP and Ch IP-PCR results confirmed that CDK19 could directly bind to p53 to the promoter region of p21 and thus affected the transcription process of p21.This indicates that the significantly reduced proliferation and self-renewal abilities of mouse bone marrow HSCs after CDK19 knockout may be attributed to the increased expression of p21 after activation of the p53 pathway.3.4 Treatment with p53 inhibitor（Pifithrin-β,PFTβ）significantly reduced the expression levels of p53 and p21 in CDK19-/-HSCs,and significantly increased the number of HSCs in bone marrow,enhancing their ability to exit quiescence.More importantly,PFTβtreatment could significantly improve the defects of long-term reconstitution ability of hematopoietic stem cells caused by CDK19 deletion.These results suggest that CDK19maintains the proliferation and self-renewal abilities of HSCs by regulating the p53-p21pathway.4.CDK19 is a key molecule that preserves the proliferative ability of AML cells4.1 Database results showed that CDK19 was closely related to cell cycle progression in AML.q PCR results showed a significant increase in CDK19 expression in the leukemia cell line（HL-60 and NB-4 cells）.Senexin B could significantly inhibit the proliferative capacity of HL-60 and NB-4 cells.The above results suggest that CDK19 may play an important role in the proliferation of AML cells.4.2 q PCR results showed that there was no significant change in p53 expression levels in HL-60 and NB-4 cells after Senexin B treatment,while p21 expression levels increased significantly.Gene expression analysis found that CDK19 was not significantly correlated with with p53 expression levels in AML,but had a significant negative correlation with p21expression levels.These results suggest that CDK19 can regulate the proliferation of AML cells through the p53-p21 pathway.In conclusion,through the systematic study of the hematopoietic effect of CDK19under homeostatic and stress conditions,this study revealed that CDK19 is a key molecule that participates in the regulation of cell cycle in HSCs and AML cells,CDK19 can promote the recovery of bone marrow hematopoietic function under radiation stress conditions.This study not only enriches the understanding of hematopoietic stem cell homeostatic regulation network,but also provides certain guidance for the treatment of bone marrow hematopoietic damage caused by IR and acute myeloid leukemia. |
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