Effects Of RIP3 And RIP1 Knockout On Autophagy In Mouse Leukemia Cells

Acute myeloid leukemia(AML)is an aggressive hematopoietic tumor that originates from myeloid progenitor cells,which are malignantly transformed from hematopoietic progenitor cells at different stages during the differentiation and development of normal myeloid cells.The most common acute leukemia in adults is AML,and although the rate of complete response after conventional chemotherapy is high,frequent relapses due to relapse-induced leukemia cells(RIC)resistant to chemotherapy lead to frequent relapses and a poor prognosis.Autophagy is a major regulator of cellular metabolism and is involved in maintaining cancer status,metastasis,and antitumor therapy.Recent studies have shown that AML cells exhibit metabolic changes after chemotherapy or targeted therapy.However,autophagy plays a role in tumor suppression or promotion in AML remains controversial.In AML,autophagy has been shown to support cell proliferation in vitro and leukemia progression in vivo.Recent studies have shown that mitochondrial autophagy is necessary for leukemia stem cell function and survival,highlighting the prominent role of this selective autophagy in the occurrence and development of leukemia.In addition,autophagy in AML maintains fatty acid oxidation through lipid phagocytosis to support mitochondrial oxidative phosphorylation,a hallmark of chemotherapy-resistant cells.In the clinical treatment of AML and other cancers,autophagy may have cytoprotective or cytotoxicity,depending on the drug used.Receptor interacting serine/threonine protein kinase(RIP)family is an important cellular stress regulatory molecule,mainly involved in regulating apoptosis and necrotizing apoptosis of cells,of which RIP1 and RIP3 have been studied more in recent years.Studies have shown that the binding between RIP1 and RIP3 is a key component of the signal transduction pathway,which can be induced by TNF to form RIP1,RIP3,mixed lineage kinase domain-like protein(MLKL)complexes in the absence of caspase inhibitors,which play an important role in the development of AML cells.There are currently reports of a link between RIP3 and autophagy,but further research is needed.This project takes MLL-AF9 AML mouse leukemia cells as the research object,and studies whether RIP3 and RIP1 knockout affect the autophagy of mouse leukemia cells and their mechanism of action,which provides a new theoretical basis and experimental basis for in-depth understanding of the development of autophagy in AML and the mechanism of drug resistance.The research mainly includes two parts:1.Explore whether RIP3 and RIP1 knockout affect autophagy in mouse leukemia cells:We determined the relationship between RIP3,RIP1 and mouse leukemia cell autophagy by detecting WT,RIP3^-/-,RIP1^-/-mouse leukemia cell resistance to chemotherapy drugs,the basal autophagy level of cells,and the changes in autophagy levels of cells after treatment with chemotherapy drugs and the localization of LC3B changes in cells at the cellular level.The MTT results showed that the resistance of cells to the detected chemotherapeutic drugs increased after RIP3 knockout,while the cells knocked out by RIP1 were only more resistant to some chemotherapeutic drugs.Western Blot(WB)results showed that WT,RIP3^-/-,RIP1^-/-mouse leukemia cells all had higher autophagy flux and were further elevated compared to WT,RIP3,or RIP1 knockout.After treating cells with different concentrations of chemotherapeutic agents(DNR,Ara-C,CPT)or treating cells with the same concentration of chemotherapeutic drugs DNR for different periods of time,the WB results showed that after treatment with chemotherapeutic agents,compared with WT and RIP1^-/-cells,the levels of p62 and LC3-II/LC3-I increased and were inversely proportional to the concentration or time.Dual fluorescent autophagy detection showed that RIP3^-/-was mostly late autophagy compared to WT,and the autophagy flow was not suppressed,while RIP1^-/-cells were early autophagy,and the autophagy flow was slightly inhibited.2.Explore the specific mechanisms by which RIP3 and RIP1 regulate autophagy in mouse leukemia cells:We explore the mechanism of RIP3 and RIP1 regulating autophagy in mice leukemia by detecting cell proliferation rate,cell DNA damage level,changes in THE LEVEL of RIP3 and RIP1 proteins during chemotherapy drug treatment,and the interaction of RIP3,RIP1 and p62 proteins.Immunofluorescence showed that WT,RIP3^-/-,RIP1^-/-mouse leukemia cells had high levels of DNA damage compared to c-Kit⁺cells.After treating WT,RIP3^-/-,RIP1^-/-mouse leukemia cells with chemotherapy drug DNR(100n M),WB detected RIP3 and RIP1 expression levels,and the results showed that the medication time increased,and the expression of RIP3and RIP1 proteins also gradually increased.The Co-IP results showed that both RIP3and RIP1 interact with the p62 protein.In summary,three conclusions are drawn:1.MLL-AF9 AML mouse leukemia cells have too much DNA damage due to abnormal proliferation rates,which causes the cells themselves to have a high level of autophagy.2.RIP3 can bind p62 protein to inhibit mouse leukemia cells from early autophagy to late autophagy,RIP1 can bind p62 protein to promote mouse leukemia cells from early autophagy to late autophagy,and RIP3 expression depends on RIP1.3.RIP3 and RIP1 may be involved in the autophagy transcriptional regulatory pathway mediated by DNA damage,and the specific mechanism needs to be further explored.