| Acute myeloid leukemia(AML)is a hematopoietic malignancy and characterized by the clonal proliferation of myeloblasts.It is the most common form of acute leukemia among adults in China.The incidence rate increases with age.Despite the emergence of new therapeutic techniques such as immunotherapy and molecular targeted therapy based on traditional chemoradiotherapy and stem cell transplantation programs in recent years,the survival and prognosis of patients with AML have been improved to some extent,but the 5-year overall survival rate of AML is about 30%-40% while the relapsed and refractory patients are less than 15%,and eventually develop chemotherapy resistance,so It is necessary to find new mechanisms to explore how leukemia cells can escape chemotherapy drugs and the Immune surveillance.At present,the basic principle of anti-cancer strategy is to eliminate tumor cells by inducing apoptosis of tumor cells by high-dose radiotherapy and chemotherapy.Recent studies have found that chemotherapeutic drugs can also induce senescence of tumor cells,Cellular senescence was initially described as a cell-autonomous tumor suppressor mechanism that triggers an irreversible cell cycle arrest that prevents the proliferation of damaged cells at risk of neoplastic transformation.However,senescenct tumor cells may be reversible and may be induced to obtain stem cell-like activity.Unlike apoptotic cells,senescent cells remain viable for long periods of time and are highly bioactive.It can affect the tumor microenvironment and adjacent cells by secreting extracellular matrix degrading enzymes,cytokines,and chemokines,which collectively constitute the senescence-associated secretory phenotype(SASP)and contribute to chronic inflammation and tumorigenesis.Another key function of the SASP is to attract immune cells,which in turn can orchestrate the elimination of damaged and senescent cells,but when the senescent cells fail to properly clear and continue to accumulate,the secretory phenotype may induce the surrounding immune cells transform to immunosuppressed phenotype,which promote the immune escape of tumor cells.All of these effects may contribute to tumor recurrence,metastasis,and no response to chemotherapy,targeting,and immunotherapy.Therefore,we can explore tumor recurrence and drug resistance from the perspective of senescence and apoptosis.The "SENEX" gene,which plays an important role in the signal transduction pathways that control cell cycle and senescence and apoptosis by regulating the transcription of some related genes,was successfully cloned in 2004.It is a novel gene encoding a kind of Ras superfamily protein named as ARHGAP18.Paul R.Coleman et al.first discovered that SENEX gene can induce TNF-?-induced apoptosis of human vascular endothelial cells by activation of p16INK4A/Rb pathway,but does not affect telomere length.And there is no replicative aging gene profile in senescent endothelial cells..Moreover,our previous study has also showed that SENEX gene expression was up-regulated in regulatory T cells(Tregs)of aged bladder cancer patients and is associated with the ability to protect Treg cells from H2O2-induced apoptosis.However,it is unclear whether the SENEX gene is expressed in patients with acute myeloid leukemia and whether it plays a role in inducing senescence and anti-apoptosis of leukemia cells.A total of 67 patients with adult AML were enrolled in our study,including 21 newly diagnosed patients,27 complete remission ones,and 19 patients who relapsed after remission.At the same time,12 age-and gender-matched iron deficiency anemia adults without tumor or immune disease were selected as controls.Then we explored the following issues: Firstly,the expression of SENEX in bone marrow mononuclear cells of each group;Secondly,the relationship between SENEX and clinicopathological parameters and therapeutic effects.Thirdly,The expression of SENEX was further investigated in vitro to study the changes of senescence and apoptosis of AML cell lines at different time points.Finally,a patient with recurrent AML was followed up and his SENEX gene,senescence and apoptosis were observed during chemotherapy.The specific experimental methods and results are as follows:(1)Firstly,We detect the expression of SENEX gene and protein in bone marrow mononuclear cells in different stages of AML patients and control group by Real-time PCR and western blot.The results showed thatthat compared with the control group(0.92±0.51),The expression level of SENEX in the bone marrow of newly diagnosed AML patients(4.06±2.72)was significantly increased.At the same time,we compared the expression of SENEX gene and protein in bone marrow mononuclear cells of AML patients at different stages of initial diagnosis,recurrence and remission.The results showed that the expression of SENEX was elevated at the time of initial diagnosis.After a period of treatment,the level of SENEX also decreased when the patient achieved complete remission(2.52±1.83),and then,when the disease relapsed,the expression of SENEX(3.27±2.54)increased again.The same trend was observed in the protein levels.It can be seen that there is a high expression of SENEX in AML patients and is closely related to the progression of AML disease;(2)Secondly,by studying the relationship between the expression of SENEX gene and the clinical features of AML patients,it is shown that the expression of SENEX is related to the percentage of myeloid leukemia cells,M3 subtype and whether remission can be achieved after the first induction,but not related to age,gender and white blood cell count.At the same time,the expression level of SENEX gene m RNA in AML patients is positively correlated with bone marrow blast cells.It is speculated that SENEX may be used as an indicator for predicting efficacy and prognosis;(3)In order to further study the function of SENEX,We successfully induced senescence in NB4 cell line cultured in vitro under the stress of cytotoxic drug cytarabine(10?M),and then detected the expression of SENEX gene m RNA by QRT-PCR.The results showed that compared with the PBS control group(1.04±0.2),the expression of SENEX m RNA was significantly increased in the low-dose Ara-c-induced group(8.50+1.4,P<0.01).It is suggested that low-dose Ara-C can induce up-regulation of SENEX gene expression in NB4 cell lines.While transferring NC and SENEX-Si RNA into NB4 cells using liposome Lipofectainin?2000.Compared with the NC group(2.78±0.9),the m RNA level of SENEX gene had a decrease in the SENEX-Si RNA group(1.36±0.6).Under the pressure of low-dose Ara-C,the level of SENEX m RNA in SENEX-Si RNA+Ara-C group(1.12±0.5)was significantly lower than NC+Ara-C group(6.24±1.1)and Ara-C group(8.50±1.4).By observing the change in the proportion of senescent cells after 12 hours of Ara-C treatment,it was found that the number of senescent cells was significantly reduced in the SENEX silenced group.These results suggest that small doses of Ara-C can induce senescence of NB4 cells in vitro,and the expression level of SENEX m RNA in senescent cells was significantly increased.Secondly,silencing SENEX expression significantly affects the premature senescence induced by low-dose Ara-C.(4)In order to study the changes of cell senescence and apoptosis caused by differential expression of SENEX,we studied the proportion of senescence and apoptotic cells in leukemia NB4 cell line induced by low-dose cytarabine(10u M)in vitro after 12 and 48 hours by ?-galactosidase staining and Annexin V/PI double staining.The results showed that the SENEX natural expressing cells had a high proportion of senescence in 12 hours and a low proportion of apoptosis in 48 hours,After functional si RNA inhibited the expression of SENEX gene,the SENEX silencing-down cells had a low proportion of senescence in 12 hours and a high proportion of apoptosis in 48 hours.Therefore,it is speculated that the SENEX gene can play a role in protecting leukemia cells by inducing senescence of NB4 cells and antagonizing apoptosis.(5)Using the same experimental method,we observed the change of cell senescence,apoptosis,and SENEX gene expression in one recurrent AML patient before and after low-dose chemotherapy.The results showed that up to 52% of peripheral blood mononuclear cells showed typical senescence changes after 2 days of pretreatment with 100 mg Ara-C.At the same time,the percentage of early and late apoptotic cells in peripheral blood mononuclear cells increased significantly compared with pre-treatment,and SENEX m RNA levels were also up-regulated by 4 fold(5.2% vs.1.0%),suggesting that SENEX can also promote senescence in vivo.What's more,the patient's SENEX gene expression was found to be at a low level after reaching remission,and no senescent cells were found in the peripheral blood and bone marrow during the follow-up.In summary,this study demonstrates that SENEX is significantly up-regulated in both newly diagnosed and relapsed AML patients,but significantly lower in patients with complete remission after treatment.The expression level of SENEX gene was different in different subtypes of AML patients,among which M3 was the highest.Secondly,we successfully induced senescence of NB4 cells in M3 cell line using low-dose Ara-C in vitro and demonstrated a significant increase of SENEX expression in senescent cells.The proportion of senescent cells was significantly decreased when the SENEX was silenced,suggesting that SENEX is a key molecule in the induction of senescence of NB4 cells by low-dose Ara-C;Further studies have found that the effect of SENEX may be related to stress-induced stress premature aging(SIPS)-mediated apoptosis resistance of AML cells.ALL of these results suggest that SENEX may be a new therapeutic target for AML treatment,and the exact molecular mechanism of SENEX gene in the development of AML remains to be further studied. |
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