Study On The Effect Of RIP3-deficiency On The Sensitivity Of Leukemia Cells To Daunorubicin

RIP3(receptor-interacting serine/threonine protein kinases 3)is a member of serine/threonine receptor mediated family.In addition to the RIP kinase domain specific to the RIP kinase family,RIP3 has a unique C-terminal caspase activation and recruitment domain.This domain can bind to RIP1 and induce programmed necrosis.Studies have reported that RIP3 plays an important role in the development and progression of leukemia.Programmed necrosis was previously considered to be an alternative process of cell death when apoptosis was inhibited.At present,programmed necrosis is considered to play an important role in regulating various physiological processes.It is worth noting that it mediates a variety of human diseases,such as ischemic brain damage,immune system disorders and cancer.Acute myeloid leukemia(AML)is a heterogeneous hematological malignant tumor characterized by immature clonal myeloid cell proliferation and abnormal differentiation.At present,leukemia is mainly treated with chemotherapeutic drugs.The poor prognosis of AML,especially in the elderly,is mainly related to leukemia recurrence and non-response to initial treatment.In many leukemia cases,some cells will survive,expand and erupt in chemotherapy drugs,have a low long-term survival rate,a high recurrence rate,and are often resistant to existing treatments at high recurrence rates,and resistance remains a serious problem.The regulation of apoptosis signal pathway plays an important role in overcoming drug resistance of leukemic stem cells.With the in-depth study of the mechanism of drug resistance in leukemia,autophagy has been found to play a vital role in the drug resistance of leukemia to chemotherapeutic drugs.Based on this,this research aims to investigate whether RIP3 deletion can affect the sensitivity and mechanism of MLL-AF9 AML leukemia cells to chemotherapeutic drugs.Objective:Based on this,taking MLL-AF9 AML cells as the research object,the research of this subject mainly includes two parts:1.To explore whether RIP3-deficiency can affect the sensitivity of MLL-AF9 AML cells to chemotherapeutic drugs DNR;2.To study the regulatory role of RIP3-deficiency on growth and development in MLL-AF9 AML cells facing the stress response of serum starvation.Methods:1.MTT colorimetry and flow cytometry were used to detect the effects of RIP3 loss on cell proliferation,cell cycle or cell differentiation after 48h of treatment with and without the chemotherapy or 12h of starvation stress.2.Annexin V/PI staining and western-blot analysis were used to detect the apoptosis,LC3 and Bcl-2 protein expression levels of cells treated with and without DNR for 48 h or 12 h after starvation stress.3.Cell morphological changes and LC3 protein expression were observed by Giemsa staining and immunofluorescence staining.4.The expression of genes related to poor prognosis was detected by qRT-PCR.Results:1.After 48 hours of treatment with different concentration gradients of DNR,the cell mortality showed an increasing trend with the increase of concentration,while the proliferation rate of RIP3 deficient cells was accelerated compared with the WT group and there was no significant difference between GSK'872 group and WT group;2.After treatment with 10 nM DNR for 24 h,most cells were blocked in the S/G2 phase,the apoptosis level of RIP3-deficient cells was decreased,the expression levels of LC3 and Bcl-2 was up-regulated,and the expression of related genes was decreased.3.After 12 h of serum starvation treatment,the RIP3-deficient cells had not only faster cell proliferation and integral cell membrane structure than the WT group and the cytoplasm appeared similar to the autophagic vacuole structure,but also the percentage of CD117 positive cells and Annexin V~+PI~-cells was decreased,and the LC3 expression was increased significantly.Conclusions:1.RIP3-deficiency may promote the proliferation of leukemia cells by triggering the activation of survival signaling pathways,which in turn leads to a reduction in the sensitivity of leukemia cells to DNR,regardless of its cell cycle.2.RIP3 deletion can regulate the stress response of MLL-AF9 leukemia cells to serum starvation.3.The results of this study suggest that RIP3 deletion activates the important signaling molecules Bcl-2 and LC3 to mediate cell proliferation,cell apoptosis and cell autophagy,which are involved in the protection of leukemia cells and may be an important molecular mechanism for AML cells to resist DNR,and the specific mechanism of this resistance is in urgent need of further exploration.