The Study On The Role And Michanism Of MiR-155 And Its Target SHIP1 In The Pathogenesis Of Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a common type of leukemia. At present, only about 40% of the younger AML and about 10% of the elderly AML can achieve long-term survival by conventional chemotherapy. In order to reduce the conventional therapy-related side effects and improve the overall survival of patients, we should further explore the pathogenesis of AML and new therapeutic strategies.The pathogenesis of AML is a multi-step process. Because of the gene regulation disorders, the proliferation, apoptosis and differentiation of cells are out of control, eventually leading to the malignant transformation of hematopoietic stem and progenitor cells. Phosphatei-dylinositol 3 kinase /serine-threonine kinase (PI3K/AKT) signal transduction pathway is one of important cell signal pathways. Excessive activation of this pathway will affect the downstream effector molecules, result in the uncontrolled proliferation, inhibition of apoptosis, and promote the development of malignant diseases. PI3K/AKT pathway plays an important role in 50%-70% of AML, but the mechanism is still remained unknown.The SH2 domain-containing inositol 5’-phosphatase 1 (SHIP1) is a class of phosphatidylinositol phosphatase (PIPase) that is important in the negative regulation of the proliferation and survival of hematopoietic cells through its negative regulation of the PI3K/AKT signaling pathway. Recent studies have shown that the inactivation of SHIP1 could play a central role in some hematologic malignancies. Inactivation of point mutations of the SHIP1 gene and reduction of SHIP1 activity have been observed in patients with acute myeloid leukemia (AML), implicating a possible tumor suppressor function of SHIP1 in the pathogenesis of AML. However, it appears that SHIP1 gene mutations are an uncommon cause of reduction in SHIP1 activity in AML Several other possible explanations that could account for the loss of SHIP1 function include translational repression by post-transcription of microRNA (miRNA), epigenetic modification, decreased transcription and increased protein breakdown.MicroRNAs are a newly discovered class of short, noncoding RNA species, which may serve as master switches in gene networks. MiR-155 was shown to bind to the 3’UTR of SHIP1 mRNA and inhibit its translation. SHIP1 is expressed in the same cell types that express miR-155, and it plays an opposing role in many cases. Some studies demonstrated a strong correlation between myeloproliferative disorders (MPDs) caused by miR-155 expression and specific knockdown of SHIP1.It was demonstrated that SHIP1 is a primary target of miR-155 in B cells, with high levels of miR-155 and reduced expression of SHIP1 linked to the development of acute lymphoblastic leukemia in mice. Levels of miR-155 were also found to be significantly increased in human patients with diffuse large B cell lymphoma or NK/T cell leukemia with decreased SHIP1 expression.Further investigation is needed to evaluate the role of miR155 and SHIP1 in the pathogenesis of AML. Up to now, there are no any reports about the mechanisms of miR-155 and its target SHIP1 on the development of AML in the world.In this study, we examined the levels of SHIP1 protein and miR-155 in samples of patients with AML and in AML cell lines. In addition, we investigated cell proliferation, apoptosis and expression of SHIP1/PI3K/AKT pathway molecules in cell lines of THP-1 and U937 after miR-155 inhibitor or mimics were transfected. The study has laid a foundation for further study of miR-155 and SHIP1 in the pathogenesis of AML, so as to provide an important basis for the explanation of the role of miRNAs and miRNA-targeted treatment of AML.Part I. miR-155 and SHIP1 expressions in patients with acute myeloid leukemia and cell linesObjective:SHIP1 encoded by the INPP5D gene, is a negative regulator of PI3K/AKT signal transduction pathway of cells. PI3K/AKT signal pathway play an important role in the pathogenesis of 50%-70% of AML, but the specific mechanism keeps unknown. Studies have shown that SHIP1 is one of the targets of miR-155. miR-155 has been confirmed the increased expression in some types of AML. The purpose of this study was to mearure the gene expression of miR-155 and SHIP1 in AML patients and leukemia cell lines.Methods:Quantitative PCR was used to detect gene expressions of miR-155 and SHIP1 at mRNA levels in peripheral blood or marrow samples from 30 AML patients, and bone marrow samples from age-matched healthy people were used as controls. Expressions of miR-155 and SHIP1 were also measured in cell lines by quantitative PCR. SHIP1 expression at protein level in AML patients and cell lines was detected by Western blot. According to the expression level of SHIP1 protein and AML subtypes, patients were divided into 1 groups and 2 groups.Clinical features and complete remission (CR) rates were compared between the two groups. The prognostic factors influencing CR were analyzed by multivariable analysis.Results:Compared with those from healthy controls, expressions of SHIP1 were significantly decreased in AML patients (P<0.05), but there were no differences found in its expression among AML subtypes. Significantly decreased SHIP1 protein levels were found in 15 patients with AML-M4/M5, which were combined with increased expression of miR-155 (P all<0.05). miR-155 was an independent prognostic factor in the normal karyotype of AML.miR-155 was lower expressed in K562 cell lines, while relatively highly expressed in both THP-1 and U937 cell lines, especially in THP-1. Expression of SHIP 1 was found highly expressed in U937 cells.Conclusion:In patients with AML-M4/M5, miR-155 expression was elevated while SHIP1 expression at the protein level was decreased. miR-155 may be involved in the pathogenesis of AML by targeting SHIP1. miR-155 expression highly expressed in both U937 and THP-1, indicating these two cell lines can be used for further experiment to explore the effect of miR-155 on the pathogenesis AMLPart II:miR-155 overexpression regulates SHIP1/PI3K/AKT pathway and poliferation and apoptosis in U937 cellObjective:miR-155 is mainly expressed in hematopoietic cells, playing a role as an oncogene in malignant diseases. SHEP1 mainly expressed in hematopoietic cells, acted as a tumor suppressor and a negative regulation factor of PI3K/AKT signaling pathway. SHIP1 is one of the target genes of miR-155. Studies showed that expression level of miR-155 increased and expression level of SHIP1 decreased in patients with some subtypes AML. In the present study, miR-155 is overexpressed in U937 cells by transfected with miR-155 mimics. Effects of miR-155 overexpression on SHIP1 expression, PI3K/AKT pathway, proliferation and apoptosis in U937 cells will be studied. How miR-155 acts as an oncogene involved in the pathogenesis of AML is to be explored.Methods:U937 cells were divided into three groups:cells were transfected with miR-155 mimics (U937m) by using X-treme GENE siRNA Transfection Reagent, untransfected cells (U937c) and transfection control cells (U937mc). Real-time quantitative PCR was used to check transfection efficiency by measuring miR-155 gene expression. Cell proliferation was detected by CCK-8 assay and flow cytometry method was used for apoptosis analysis. SHIP1 gene expression was measured both at mRNA level by realtime-PCR and at protein level by Western blot. Protein levels of AKT and PAKT were all measured by Western blot.Results:Compared with those of U937C and U937mc, miR-155 expression was significantly higher in U937m (p<0.05). Overexpression of miR-155 led to significantly reduced apoptosis rate in U937 cells (p<0.05) and no significant increased proliferation rates at any time point (p>0.05) Overexpression of miR-155 has no effect on SHIP1 gene expression at mRNA level, but caused significantly lower SHIP1 protein levels (p<0.05), indicating there might exist the translational regulation of miR-155 on SHIP1. Overexpression of miR-155 caused no alteration in total AKT (TAKT) protein content, but significantly increased pAKT protein levels (p<0.05).Conclusion:Overexpression of miR-155 may activate PI3K/AKT signaling pathway by inhibiting SHIP1 at protein level, and inhibit apoptosis of U937 cells. miR-155 might act as an oncogene by activating PI3K/AKT pathway through targeting SHIP1, which may play an important role in the pathogenesis of AMLPart III:Effects of miR-155 inhibition on SHIP1/PI3K/AKT pathway and on proliferation and apoptosis of THP-1 cellsObjective:miR-155 is currently the most studied miRNA, and is also the first miRNA found to have carcinogenic effects. miR-155 may play a role as an oncogene targeting on SHIP1 in AML. miR-155 is a promising new target for the treatment of leukemia. In the present study, miR-155 is inhibited in THP-1 cells by transfected with miR-155inhibitor. Effects of miR-155 inhibition on SHIP1 expression, PI3K/AKT pathway, proliferation and apoptosis in THP-1 cells will be studied. The mechanism of inhibition of miR-155 on AML treatment is also to be explored.Methods:THP-1 cells were divided into three groups:cells were transfected by miR-155 inhibitor by Application X-treme GENE siRNA Transfection Reagent (THP-1I), untransfected group (THP-1C) and transfection control group (THP-1IC). Real-time quantitative PCR was used to check the transfection efficiency by measuring miR-155 gene expression. Cell proliferation was detected by CCK-8 assay and flow cytometry method was used for apoptosis analysis. SHIP1 gene expression was measured both at mRNA level by realtime-PCR and at protein level by western blot. Protein levels of AKT and pAKT were all measured by western blot.Results:Compared with THP-1 C and THP-1IC, miR-155 expression was significantly lower in THP-1I cells (p<0.05). Inhibition of miR-155 resulted in significantly increased rate of cell apoptosis rate but no significantly decreased rate of cell proliferation (p>0.05). Inhibition of miR-155 has no effect on SHIP1 gene expression at mRNA level, but caused significantly increased SHIP1 protein levels (p<0.05), indicating there might exist the translational regulation of miR-155 on SHIP1. Inhibition of miR-155 caused no alteration in total AKT (TAKT) protein content, but significantly decreased pAKT protein levels (p<0.05).Conclusion:miR-155 inhibition may promote apoptosis in THP-1 cells by inhibiting PI3K/AKT signaling pathway through increasing the SHIP1 protein content, suggesting miR-155 inhibition might be a promising strategy for anti-leukemia therapy.