Investigation About The New Functioning Mechanism Of Hoomoharringtonine In Acute Myeloid Leukemia

Part 1 Virtual screening for the protein target of homoharringtonineObjective:To screen out the potential protein target for homoharringtonineMethod:Use software including autodock,MGLtools,Python to screen the possible protein target of homoharringtonine.Use homoharringtonine as the ligand,and signal transduction molecules that have been reported to related with homoharringtonine functioning as the receptor,then use molecular docking models to simulate the binding between the ligand and the receptor,and screen according to the binding energy of the conformations.Result:(1)We docked 30 proteins with homoharringtonine,amongst them smad3 has the lowest binding energy.(2)According to the virtual binding conformation,HHT might associate with the MH2 domain of smad3.Conclusion:smad3 is the potential target protein for HHT.Part 2:Investigation of the relationship between homoharringtonine and smad3 as well as TGF-? pathway in AML cell lineObjective:To verify the conclusions of part 1,to investigate the relationship between HHT and smad3,HHT and TGF-? pathway.Method:Use MTT to detect the inhibition of HHT on AML cell proliferation,and the effects of HHT on U937 cells before and after transfection of smad3-shRNA.Use western blot to detect the protein level changes of smad3 upon HHT functioning,to detect the nuclear protein levels of smad2/3 and smad4,to detect the level of cell-cycle related proteins after HHT functioning;Use immunofluorescence to detect the translocation of smad4 within the cell;Use flowcytometry to detect cell apoptosis and cell cycle arrest;Use lentivirus transfection system to transfect smad3-shRNA into U937 cells;Use colony forming test to detect the influence of HHT on colony-forming abilities of U93 7 cells before and after transfection.For data analysis,use t test for single variant evaluation between,two groups,for comparasion amongst groups,use one-way ANOVA,take p<0.05 as being significant.Results:(1)HHT can promote smad3 ser423/425 phosphorylation.(2)HHT can promote smad4 translocation in to nucleus,and increase smad2/3 protein level in the nucleus.(3)HHT can induce cell cycle arrest,not apoptosis in U937 cells,cells are arrested in G1 phase,and there are corresponding protein level changes in c-myc,p15 and CDK4/6.(4)smad3-shRNA can significantly reduce the protein level of smad3.After transfection,IC50 of HHT increases,G1 phase arrest decreases upon HHT functioning.And more colonies can be formed after transfection when exposed to the same concentration of HHT.Conclusion:(1)HHT can activate smad3 through ser423/425 phosphorylation.(2)HHT can activate TGF-? pathway through smad3.(3)The behavior of HHT to induce cell cycle arrest and the change of the corresponding proteins fits that of the activation of TGF-?.(4)Transfection of smad3-shRNA can decrease the sensitivity of U937 cell to HHT,in the manner of less cell cycle arrest and more colony formation.(5)Smad3 and TGF-? pathway are the potential targets of HHT.Part 3 Investigation of the relationship between smad3 expression level and the clinical behavior of AML patientsObjective:To detective smad3 levels in de novo AML samples,and investigate the relationship between the smad3 expression level and the patients' clinical behavior.Method:Collect the mononucleated cells from the patients'bone marrow using lymphocyte separation solution,and extract 'RNA.Use Real-time quantitative PCR to detect the mRNA level of smad3,use PCR and Sanger sequencing to detect gene mutation.Use Kaplan-meier method to analyze survival conditions,and Cox regression model to investigate the relationship between smad3 and other factors.Results:(1)Patients with high level of smad3 has better OS and EFS.(2)In Cox model,expression level of smad3 is closely related with OS.Conclusion:Smad3 is the independent prognostic factor for the OS of de novo AML patients.