| Background Acute myeloid leukemia（AML）is a common hematological malignancy,accounting for 80%-90% of adult acute leukemia.The incidence of AML is about 4.3/100,000 and increases with age,which exceeds 20.1/100,000 in the elderly over the age of 65.In AML,the complete remission rate of initial therapy is 60-80%,but the refractory rate is as high as 50%-70%,and the 5-year overall survival rate is only 30%-40%,leading to the most deadly hematological malignancy.In addition to the special subtype of acute promyelocytic leukemia with retinoic acid with arsenite induction differentiation therapy,chemotherapy is still currently main treatment in the other subtypes of AML subtypes.The qualified can choose with molecular target therapy or sequential allogeneic hematopoietic stem cell transplantation.Most of the remaining AML patients have limited remission depth,and minimal residual disease（MRD）that has not been cleared in the body which will eventually lead to disease recurrence and death,except for the part of the AML treated with allogeneic hematopoietic stem cell transplantation.It is a key issue that urgently to be solved in current basic scientific research and practical clinical work.Cellular senescence is a response to various non-lethal internal and external injuries that result in a fairly stable and static state after cell removed from its normal cell cycle and lost its ability to proliferate.Among them,stress induced-premature senescence refers to the telomere independent cellular senescence induced in advance by the stimulation of various stress factors.Different from the telomere dependent replicative senescence,it is considered to be the protective response mechanism of the body to maintain the stability of the genome and an important natural barrier to prevent the occurrence and development of tumors on the early view.But in recent years,more and more studies show that many kinds of tumor cells can get into the "old but not die" temporarily "hibernation" state to resistance to chemotherapy and mediate relapse under chemotherapy drugs,through irritability premature aging of this mechanism.This process is in-depended on any molecular genetic changes.The word SENEX originates from Latin "old man",so "SENEX gene" is also known as "senescent gene".It is named ARHGAP18 in Refseq system,located at the long arm of human chromosome 6（6q22.33）,with a total base length of 2901 bp.It encodes protein containing 663 amino acid residues with the molecular weight of 75 KD,which is widely distributed in various tissues and organs of human body.It can function as a "molecular switch" for quickly sense cell stress to activate cells signal pathway,in order to regulate the expression of many key genes and maintain cell survival.Studies have found that over-expression of SENEX gene can induce in vascular endothelial cells,regulatory T cells and lymphoma cells to typical morphological characteristics of cellular senescence,as well as cell cycle arrest and senescence related secretion phenotypes,so as to resist apoptosis induced by external stress factors.Nevertheless,the expression,function and mechanism of SENEX gene in AML have not been reported by other research groups.Also few studies have reported the occurrence and role of stress induced-premature senescence in AML.Our research group previously found that the expression level of SENEX gene in AML patients increased,especially when there was no remission after chemotherapy,suggesting that SENEX gene might be involved in AML drug resistance and recurrence.This study intends to further explore and analyze the role of SENEX gene in AML recurrence and its specific mechanism from the approach of stress-induced premature senescence to provide experimental basis for elucidating the relapse mechanism of AML.Methods Clinical specimen experiment: Collection of 16 AML patients with newly diagnosed,20 AML patients with remission,18 AML patients with refractory/recurrent and the control group of 19 patients with iron deficiency anemia,peripheral blood and bone marrow samples were collected from each group,at the same time,peripheral blood samples were collected from some of the above-mentioned patients with relapsed and refractory AML undergoing chemotherapy.Cell smears of AML patients were stained by Wright-Giemsa staining to observe the morphological characteristics of leukemia cells and their morphological characteristics under the stress of chemotherapy.Direct morphological staining of β-Galactosidase（SA-β-Gal）and combined flow cytometry（FCM）were used to analyze the proportion of AML leukemia cells and their senescence under the stress of chemotherapy.The expression of RNA-seq was detected by single cell sequencing,and the expression of senescent genes in leukemia cell subsets of AML patients was analyzed.Real-time quantitative PCR（q PCR）and Western Blot were used to detect the m RNA and protein of SENEX gene in AML patients,and the expression characteristics of SENEX gene at the transcription level and translation level and its correlation with relapse were analyzed.In vitro cell line experiments: The cell model of stress-induced premature senescence was established by cytarabine in vitro induction of AML cell line KG-1a,and then the morphological changes of cells were observed by Wright-Giemsa staining,the changes of cell senescence ratio were analyzed by SA-β-Gal direct morphological staining and FCM detection,the changes of cell proliferation rate were analyzed by CCK-8 detection,and the changes of cell apoptosis ratio were analyzed by FCM detection.Cell cycle changes were analyzed by FCM,cytokine-related secretion phenotypes were analyzed by CBA assay,SENEX gene transcription level expression was detected by q PCR,SENEX gene translation level expression was detected by Western Blot.Then SENEX gene in KG-1a cell line was silenced by si RNA lentivirus stable transfection technology,and the KG-1a cell were induced in vitro by cytarabine,and the changes in morphology,proliferation,senescence,apoptosis and cycle were detected and analyzed after SENEX gene silencing.Results Clinical specimen experiment: Experimental analysis of patients with clinically relapsed and refractory AML showed that,compared with newly diagnosed AML patients and patients in remission,the volume of leukemia cells remaining after chemotherapy in patients with relapsed and refractory AML was significantly increased by WrightGiemsa staining.In addition,both morphological direct staining and FCM analysis showed that the positive proportion of senescence-specific SA-β-Gal staining was significantly increased,especially in patients with relapsed and refractory AML during chemotherapy.We collected bone marrow samples from patients with relapsed and refractory AML after chemotherapy to fully verify the high proportion of stressedinduced premature senescence and analyzed the expression of RNA-seq by single cell sequencing.The results showed that the expression of senescence-related genes in leukemia cell subsets was significantly up-regulated compared with other cell subsets.We also found that SENEX gene expression in newly diagnosed and refractory AML patients was significantly increased at the transcriptional level and translation level compared with the control group and the remission group of AML patients with iron deficiency anemia.In vitro cell line experiments: we used AML cell line KG-1a for in vitro induction experiments,and the results showed that KG-1a cell were treated with 10μM cytarabine for 3 consecutive days and 2 hours a day for 6 days without drug intervention.Compared with 0 day basal line without drug intervention,the number of KG-1a cell and the proliferation rate were significantly reduced after cytarabine treatment,apoptosis rate increased significantly.Moreover,the change of the 6 days experimental group was more obvious than that of the 3 days experimental group.However,whether cytarabine was treated for 3 days or 6 days,some leukemia cells were still able to survive drug resistance and showed a stress-induced premature senescence phenotype,its volume increases in morphology,morphology and flow cytometry SA-β-Gal senescence staining positive percentage were significantly increased.At the same time,cell cycle analysis was performed on KG-1a cell induced by cytarabine above,and the results showed typical cycle arrest state.At the same time,cytokine detection was performed,and the results showed that senescence-related IL-8/IL-6/IL-1β were significantly up-regulated compared with 0 base-line data.Next,we silenced SENEX gene in KG-1a cell lines by si RNA lentivirus stable transfection technology.It was found that when SENEX gene was silenced in KG-1a cell and then induced by cytarabine,the results showed that the control group without SENEX gene silencing KG-1a cell had a high proportion of senescence but a low proportion of apoptosis.After relieving cytarabine stress,cell proliferation could be restored and the lethal effects of chemotherapy drugs could be eliminated.However,the SENEX gene silenced experimental group KG-1a cell had a low senescence rate but a high apoptosis rate,and the cell proliferation could not be restored after the release of cytarabine stress and continued culture,and eventually died from the lethal killing of chemotherapy drugs.Conclusion AML patients with relapse and refractory state had a high proportion of stress-induced premature senescence and its related cycle arrest and secretory phenotype,accompanied by a significant increase in SENEX gene expression level.SENEX gene was highly expressed when AML cells were subjected to chemotherapeutic drug stress in vitro and promoted stress-induced premature senescence mediated drug resistance and relapse of AML cell. |
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