Study On Effects Of EPS8 On The Proliferation, Invasion And Drug Sensitivity Of Chronic Myeloid Leukemia Cells In Vitro And In Vivo

Chronic myeloid leukemia(CML)is a myeoloproliferative neoplasm,and its incidence accounts 15%of adults which are attacked by leukemia.The genetic feature of CML is the BCR-ABL fusion gene,which plays an important role in occurrence and development of CML because of its high tyrosine kinase activity.As the result,the BCR-ABL gene becomes the specific target of CML therapy.The tyrosine kinase inhibitors(TKIs)opens up a new era of CML targeted therapy.However,with the accumulation of clinical application,drug resistance of TKIs is increasing.Therefore,it is a trend to find new anti-tumor targets and explore new targets combination with TKIs for CML molecular targeted therapy.Epidermal growth factor receptor pathway substrate 8(EPS8)is a 97 kDa protein that is tyrosine phosphorylated following stimulation of receptor tyrosine kinases.Multiple studies have shown that as a tumor related antigen,EPS8 is known to be involved in many human solid cancers,and its overexpression level plays an important role in the development of tumor and the invasion and migration of tumor cells.In recent years,we have found that EPS8 is highly expressed in a variety of malignant hematologic tumors,and is associated with poor prognosis.However,the effect of EPS 8 on malignant biological activity and Imatinib sensitivity of CML in vitro and in vivo is unclear.The current study was to examine the oncogenic function of EPS8 and its effectiveness on the Imatinib therapy in vitro and in vivo in CML by silencing or overexpressing EPS8 in CML cells to explore the new clues to CML molecular targeting treatment.The study was divided into three chapters.The aim of Chapter 1 was to investigate the effects of stable low expression or stable high expression of EPS8 on the proliferation and invasion in CML cells in vitro and in vivo.Lentivirus transfection technology was carried out to construct the K562-RNAi cell lines which existed low expression of EPS8 and the KBM5-EPS8 cell line which existed high expression of EPS8.And the level of EPS8 mRNA and protein was detected by q-PCR and western blot;The proliferation of cells after silencing or overexpressing EPS8 was detected by MTT and Trypan blue exclusion;The clone formation ability of silencing or overexpressing EPS8 was detected by Methylcellulose cloning assay;The invasion ability of silencing or overexpressing EPS8 was detected by Transwell invasion assay;The in vivo tumorigenesis ability of silencing EPS8 of CML cells were detected in Balb/c nunu mice;The CML animal models were established by injecting K562-RNAil or K562-NC cells to the tail vein of Balb/c nunu mice and were verified by Wright stain assay.In conclusion,Overexpression EPS8 can enhance the proliferation,clone formation ability and the invasion ability of CML cells in vitro.Meanwhile,silencing EPS8 can inhibit the proliferation,clone formation ability and the invasion ability of CML cells in vitro.And the xenograft tumor experiment suggested that silencing EPS 8 can inhibit the CML tumorgenesis.Overall EPS8,as a tumor related antigen,plays an important role in malignant biological activity in CML cells in vitro and in vivo and it may become a new target of CML molecular therapy.The aim of chapter 2 was to investigate effects of silencing or overexpressing EPS8 on Imatinib sensitivity in vitro and in vivo.The sensitivity of CML cells to Imatinib after silencing or overexpressing EPS8 was tested by MTT;The effectiveness of apoptosis induced by Imatinib after silencing or overexpressing EPS8 were measured by flow cytometry;After establishing the CML animal model successfully,on the fourth day after injection,the mice were next treated with imatinib at 100mg/kg once a day for two weeks(PBS was used as the untreated control).The survival time of different groups were recorded and conducted by Kaplan-Meier plot.In conclusion,EPS8 knockdown enhanced imatinib-induced cytotoxicity in vitro and in vivo in CML cells and combination EPS8 inhibition with imatinib therapy improves overall survival in human CML models.The aim of chapter 3 was to investigate effects of silencing or overexpressing EPS8 on Imatinib sensitivity and to explore the role of autophagy in it.The effect of autophagy level of silencing or overexpressing EPS8 in CML cells was carried out by western blot;The effect of autophagy level of overexpressing EPS8 in CML cells was carried out by and transmission electron microscopy;The effect of Imatinib sensitivity of silencing or overexpressing EPS8 in CML cells and the relationship between drug sensitivity and autophagy were detected by western blot;The effect of Imatinib sensitivity of silencing or overexpressing EPS 8 in CML cells and the relationship between drug sensitivity and autophagy were detected by and immunoflorescence microscopy.In conclusion,EPS8 may regulates the Imatinib sensitivity of CML cells by regulating autophagy partly.In summary,this study explored the internal mechanisms of the malignant biological activity of EPS8 and provided a novel nation that combination EPS8 inhibition with imatinib-therapy might be a potential new strategy to overcome CML drug resistance.