The Role Of Stat3/NF-?B In The Development And Drug Resistance Of MLL-r Acute Myeloid Leukemia

Leukemia is one of the diseases in the blood system,which can be understood specifically as malignant tumor in the hematopoietic system.The symptoms of leukemia are the abnormal increase of leukocytes in the blood,anemia and enlargement of the spleen and lymph nodes caused by leukocyte infiltration.Leukemia can be divided into acute leukemia and chronic leukemia according to the type of cell differentiation and the rate of disease development.Acute myeloid leukemia?AML?in acute leukemia is characterized by the rapid development of the disease,only a few months can deteriorate,the prognosis of patients is poor and easy to relapse after cure.In this study,we focused on the development of AML cells and wanted to explore a new approach to the treatment of AML.The cell selected in this study were murine fusion AML cells MLL-AF9.In the previous work of our group,the experimental results have been obtained:the knockout or mutation of the receptor interaction protein 1?RIP1?gene?lysine mutation at site 45 to alanine?can cause the reduction of clonogenic capacity of MLL-AF9 cells and the reduction of pathogenicity after transplantation in mouse leukemia cells.Based on these results,we conducted a follow-up study:1.Whether the knockout or mutation of RIP1 has an impact on the drug resistance of MLL-AF9cells to chemotherapy drugs?cytosine arabidossin and daunorubicin?;2.Whether the inhibition of signal transduction and transcriptional activator?Stat3?and nuclear transcription factor?NF-?B?has an impact on the development of drug resistance and disease in MLL-AF9 cells.We used the MTT cytotoxicity assay and the cell clone formation assay to compare the effect of the knockout or mutation of RIP1 on the resistance of MLL-AF9 cells to daunorubicin and cytosine.The experimental results showed that when RIP1 was knockout or its no.45 amino acid site was mutated,the resistance of MLL-AF9 cells to cytarabine significantly decreased.In cell clone formation experiment,when the drug concentration was fixed at 50 nM,RIP1 knock or mutation also showed the same trend as the results of MTT cytotoxicity experiments,that is,higher sensitivity to cytosine arabidosine.But in exploring RIP1 absence or mutation of MLL-AF9 cells about soft erythromycin resistance effect,we got a contrary to cytarabine resistance experiment results,namely the RIP1 absent or mutation makes the MLL-AF9 cells for soft erythromycin resistance increases significantly,the result indicates that we are in the actual treatment for the use of chemotherapy drugs to be selective.We transfected MLL-AF9 cells with I?B super repressor plasmid to inhibit NF-?B signaling pathway,and then transfected MLL-AF9 cells with lentiviral transfection to inhibit Stat3 expression with sh-RNA.For purpose of cell lines,we determined by MTT cell toxicity experiment,and the ability to clone forming experiments to explore Stat3 and NF-?B signaling pathway inhibition of the change of resistance to chemotherapy drugs,the results showed that when Stat3 and NF-?B signaling pathway inhibition,MLL-AF9 cells for cytarabine and soft erythromycin resistance decreased significantly,but when separate inhibition of Stat3 protein expression,its resistance has no obvious change.Subsequently,we compared the differences in the ability of several cells to develop disease by recording the survival status and time of the mice and the development of leukemia cells in each tissue after transplanting several cells into mice.Results the survival time of mice transplanted with Stat3^KD-migr1-MLL-AF9 cells was the longest,followed by the I?B^SR-migr1-MLL-AF9 group,followed by the Stat3^KD-I?B^SR-MLL-AF9 group,followed by the control group of WT-MLL-AF9 mice.The infiltration rates in peripheral blood,bone marrow and spleen tissues of each group were from high to low:Stat3^KD-I?B^SR-MLL-AF9,Stat3^KD-migr1-MLL-AF9,I?B^SR-migr1-MLL-AF9,WT-MLL-AF9,respectively.Which can have a conclusion for the inhibition of Stat3and NF-?B signaling pathway can effectively reduce the MLL-AF9 cells to chemotherapy drug resistance,little to prolong the life of mice,and finally organized by groups of mice in the leukemia cells infiltration rate,Stat3^KD-I?B^SR-MLL-AF9predominate group need more leukemia cells in mice to mice caused by death,said its pathogenic ability weakened.