| Acute leukemia(AL) is one of the most common form of cancers. It is characterized by symptoms of short duration, many immature cell forms in the bone marrow and/or peripheral blood, including acute myeloid leukemia(AML) and acute lymphoblastic leukemia(ALL). AML is the most common leukemia subtype in adults. As AML progresses, immature blood cells accumulate in the bone marrow, blood and organs and interfere with the production of normal blood cells, leading to fatal infection, bleeding or organ infiltration. The prognosis of survival in untreated acute forms is from several weeks to several months after diagnosis. Although there are several well-recognized risk factors for the development of AML, little is known about the etiology of most cases. Like most malignancies, there is no recognized factor common to most cases of AML. Proven or possible risk factors for AML can be categorized as genetic, environmental, and therapy-related. At this time, the proven risk factors include only radiation, benzene exposure, and chemotherapeutic agents.Initial disease remission can be achieved in60-80%of AML patients after standard induction chemotherapy regimens. Despite this success, only20-30%of patients have long-term, disease-free survival after achieving complete remission with more than50%of patients experiencing disease of relapse, the majority whom die within1year. Only about40%of younger patients and10%of older patients with this disease are alive at5years. Refractory and relapsed disease remains a major challenge in all patients.Significant progress in understanding the mechanisms leading to the development of AML has led to the identification of numerous molecular abnormalities that may be responsible for leukemogenesis. A growing understanding of the underlying molecular mechanisms of AML has led to development of novel agents, most of which selectively target leukemic cells, such as monoclonal antibodies, tyrosine kinase inhibitors, farnesyl transferase inhibitors, hypomethylating agents, histone deacetylase inhibitors, and others. For example, FLT3(Fms-like tyrosine kinase3, CD135) is a member of class III tyrosine kinase (RTKIII) receptor family and mutations of the FLT3are of major clinical relevance in AML because they commonly guide treatment decisions as independent indicators of poor prognosis. Sorafenib is one of the most extensively investigated first generation FLT3inhibitors. It has shown to specifically reduce the percentage of leukemia blasts in the peripheral blood (7.5%from81%) and the bone marrow (34%from75.5%) of AML patients with FLT3-ITD (internal tandem duplication). Acute Promyelocytic Leukemia (APL) is another example where effective targeted therapies, such as all-trans retinoic acid (ATRA) and arsenic trioxide are used and can reinstall differentiation of leukemic promyelocytes by targeting the culprit PML-RARa fusion protein. Poor outcomes in patients with AML are related to the high proportion of patients withchemorefractory disease or relapse after an initial response. The multidrug carrier P-glycoprotein (P-gp), encoded by the MDR1(ABCB1) gene, belongs to the family of membrane bound ATP-binding cassette (ABC) transporters. It is an ATP-driven efflux pump that confers multidrug resistance (MDR) to cancer cells by actively extruding a wide range of structurally unrelated chemotherapeutic compounds from the cells. Studies showed that detection of the MDR1gene product is more frequent in elderly patients and has been more strongly linked to inferior outcomes in AML. Therapeutic strategies aimed at modulating MDR1-mediated resistance may improve outcomes in patients. Given the rising incidence of AL and the identification of molecular markers in AML has greatly improved our understanding of disease pathogenesis and facilitated the discrimination of biologically and clinically distinct subgroups, novel mechanism and target-based approaches are needed to control this malignancy.Epidermal growth factor receptor pathway substrate8(Eps8) is a protein of97KDa, which is efficiently tyrosine phosphorylated by a variety of tyrosine kinases, both of the receptor(RTKs) and non-receptor type. Eps8displays a domain organization typical of a signaling molecule that includes a putative N-terminal phosphotyrosine binding(PTB) domain, a central SH3domain, and a C-terminal "effector region". Initial experiments indicated how the overexpression of Eps8conferred increased mitogenic responsiveness to EGF and increased the transforming ability of EGFR, suggesting its involvement in RTK-activated pathways leading to cell proliferation. Overexpression of Eps8is detected in many solid cancers, contributing to tumor aggressiveness and correlating with poor survival. Immunohistochemical staining for Eps8was conducted in205cases of oral squamous cell carcinoma(OSCC)collected over7years, identifying Eps8expression in186of the205cases of OSCC (91%). Univariate analysis revealed that patients with Eps8expression had significantly poorer5-year overall survival (OS) than those without it (43%vs74%, P=0.014). Clinicopathologic analysis of45patients indicated that Eps8expression was associated with parametrium invasion and lymph node metastasis, two major poor prognostic factors for early-stage cervical cancer. Kaplan-Meier analysis of cervical cancer specimens also indicated an inverse relationship between the level of Eps8and the patients’survival rate. Eps8attenuation increased cellular sensitivity to chemotherapeutic agents and thus affected the response to chemotherapy. Silencing of Eps8blocks migration and invasion in human glioblastoma cell lines suggest that Eps8might represent a useful target for the design of new drugs for the treatment of these tumors. However, the role of Eps8in hematopoietic malignancies especially in AML remains unclear. Gene expression profiling was performed on97cases of infant ALL from Children’s Oncology Group Trial P9407and initially identified Eps8was significantly associated with worse eventfree survival (EFS) in the infant cohort. The Mixed Lineage Leukemia (MLL) gene is the frequent target of chromosomal translocations and rearrangements in childhood leukemia. MLL-ABI-1fusion is common in these abnormalities. Recent studies suggest that Eps8may plays a role in leukemogenesis by translocating to MLL via e3Bl.We hypothesized that Eps8also played a crucial role in the development of AML. In this study, Eps8gene was amplified and cloned into pMD18-T to generate a recombinant plasmid pMD18-T/Eps8for the establishment of real-time quantitative PCR as standard DNA. Then this assay was used to detect the Eps8gene expression level in bone marrow samples from patients with AML and healthy volunteers. Also, Western blot was performed to confirm Eps8protein expression level in the hematological malignant cell lines. Finally, Eps8gene knockdown via Eps8specific shRNA in KGla was established to further confirm the role of Eps8in AML.Object:1.To establish a method of detecting Eps8with real-time quantitative PCR.2.To detect Eps8mRNA level in patients with AML. 3.To detect Eps8protein expression level in the hematological malignant cell lines.4.To employ Eps8shRNA medicated RNA interference approach to knockdown Eps8gene expression in KGla to confirm the function of Eps8in AML.Methods:1. Total RNA was extracted from KGla cell line and first strand cDNA was created. Eps8gene was amplified and cloned into pMD18-T to generate a recombinant plasmid pMD18-T/Eps8for the establishment of real-time quantitative PCR as standard DNA, GAPDH was used as an endogenous control.2.Standard plasmids of pMD18-T/Eps8and pMD18-T/GAPDH were diluted10-fold and tested in triplicate to establish standard curves for detection of Eps8and GAPDH. The specificity of the primer pairs was evaluated by the melting curve analysis of SYBR Green real-time PCR. To compare the sensitivity between the real-time quantitative PCR and conventional RT-PCR assay,10-fold dilutions of the recombinant plasmids were tested employing the two assays. Serial dilutions of the recombinant plasmids ranging from103to107copies/μL were amplified to assess the reproducibility of the real-time quantitative PCR. The variations in the inter-assay were assessed by testing6replicates of each concentration in a single round of the real-time quantitative PCR, and the variations in the intra-assay were assessed by repeating3rounds of the real-time quantitative PCR.3. The copy numbers of Eps8and GAPDH genes in bone marrow samples from patients with AML and healthy volunteers were extrapolated from the standard curves of Eps8and GAPDH genes.4.Total protein from was extracted from5hematological malignant cell lines for Western blot analysis.5.We designed and cloned a shRNA template targeting Eps8into a lentivirus vector containing a GFP reporter and a hU6promoter upstream of the cloning restriction sites(Agel I and EcoRI). Oligonucleotides were annealed, digested and inserted between Agel I and EcoRI restriction sites. Then infectious viral supernantants were derived by transient co-transfection of293T cells using lipofectamine2000. Infectious lentivirus vectors were harvested at48h post-transfection and the lentiviral transduction particles are titered. The Enhanced Infection Solution(ENi.S.) containing virus at a multiplicity of infection(MOI) of100was added to the human acute myeloid leukemia cell line KGla with5μg/ml polybrene. After24hours of transduction, viral media was removed and cells were selected with puromycin(2μg/ml) for14days to generate stable cell lines. The effect of Eps8knockdown in KGla was determined using Western blot.Results:1.Total RNA isolated from bone marrow samples of patients with AML, healthy volunteers and KGla cell line were identified by agarose gel electrophoresis. Three bands of28S,18S and5S were observed. The special bands from conventional PCR amplification of Eps8and GAPDH were consistent with the prospective length of amplicon sizes, which were96bp and143bp for Eps8and GAPDH, respectively. The recombinant plasmids were verified by sequencing.2.The standard curve of pMD18-T/Eps8and pMD18-T/GAPDH displayed clear linear relationship with correlation coefficient of0.999and0.999, respectively. Linear standard curves of the Eps8and GAPDH genes were obtained from102to107copies per reaction with Ct values ranging from19.389to35.857cycles and18.736to35.418cycles. Both of melting curve presented a single peak, which supported the highly specificity of primers we designed. The intra-assay coefficient of variation(CV) of the serial Eps8dilutions ranged from0.07%to0.58%, whereas GAPDH ranged from0.05%to0.59%.The CV of the Eps8and GAPDH dilutions in inter-assayranged from0.83%to1.67%and0.54%to3.80%, respectively. The detection limit of the real-time quantitative PCR was100copies, while the conventional PCR assay showed positive results only when more than103copies of the template were used. Therefore, the sensitivity of the real-time quantitative PCR is about10times greater than that of conventional PCR.3.Eps8gene can be detected in all the samples using the specific primers and probes and the data demonstrated that Eps8gene expression in patients with AML is higher than that in healthy volunteers.4.We have explored the correlation between Eps8RNA marker and the patients’ clinicopathological characteristics. The result showed that there was no significant association between the expression level of Eps8and gender, age, WBC count, platelet count, hemoglobin, presence of splenomegaly and hepatomegaly (P>0.05), but we found that the patients with AML achieved CR or not after one course of chemotherapy significantly correlated with the expression of the Eps8(P=0.021). All the patients with AML were subgrouped as either Eps8high expression group (Eps8fold change≥5) or Eps8low expression group (Eps8fold change<5)(data not show). The result showed that the CR rate of high expression group was significantly lower than that of the low expression group (P=0.024).5.We observed expression of Eps8in KGla、Raji、K562cell lines and high level of Eps8expression was visible in KG la.6.GFP expression in photomicrographs of control confirmed90%transduction efficiency at a multiplicity of Infection(MOI) of100with5μg/ml of polybrene in Enhanced Infection Solution(ENi.S.). To evaluate the silencing efficiency, transduced cells were characterized for protein expression via Western blot. KG la cells transduced with Eps8shRNA showed approximately80%reduction in protein expression relative to control. Conclusion:1.The real-time quantitative PCR assay established in this study provides sensitive, reproducible and accurate detection of Eps8gene expression and may be further used for diagnosis and predicting cancer treatment response in AML.2.Eps8gene expression in patients with AML is higher than that in healthy volunteers.3.CR rate of high expression group was significantly lower than that of the low expression group.4.High expression of Eps8was observed in KGla cell line.5.We successfully constructed the shRNA vectors targeting Eps8and confirmed that they can suppress Eps8expression in KGla cell line. |
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