The Role Of ZHX2 In The Pathogenesis And Progression Of AML And It Effects On Notch1/DLL4 Signaling Pathway

Background:Acute myeloid leukemia (AML) is a clonal hematological disease characterized by hematopoietic progenitor cells altering normal mechanisms including indefinite self-renewal, inappropriate proliferation, differentiation block and escape from apoptosis. Although improvement of AML treatment has been achieved, the long-term disease-free survival (DFS) is still less than 30%. The pathogenesis of AML is highly complex and mechanism for the involvement of pathogenesis of AML remains unknown. The zinc-fingers and homeoboxes 2 (ZHX2) is a member of a novel family which is also including ZHX1 and ZHX3 that can act as a transcriptional repressor. ZHX2 represses the promoter activity of an NF-Y-regulated gene cdc25C regulated by NF-Y, suggesting that ZHX2 partake in the regulation of NF-YA regulating genes. Several studies indicated that the low expression of ZHX2 was closely related with the process of tumorigenesis and development. Nagel et al. found that ZHX2-mediated transcriptional activation of Notch-target genes HES4 and HOXA5 while eschewing regulation via NFYA by using profiling gene expression, which suggesting an influence of ZHX2 on the Notch pathway. Several studies found the activation of the Notch pathway may indicate an unfavorable prognosis in AML. As a tumor suppressor gene, ZHX2 is of great significance in the diagnosis of hematopoietic malignancies, the expression and role of ZHX2 in AML has not yet been studied.Objective:The purpose of the present study was to detect the ZHX2 mRNA levels in non-M3 AML patients. To explore the potential roles of ZHX2 in non-M3 AML, we examined whether ZHX2 affected the proliferation and apoptotic properties of the human AML cell lines in vitro and enhance cell sensitivity to chemotherapy. Furthermore, the potential molecular mechanisms modulated by ZHX2 in leukemogenesis we also tried to find out. As its tumorigenesis role, ZHX2 could become a possible target for directing therapy.Material and methods:1. To investigate ZHX2 expression in non-M3 AML, we detected ZHX2 mRNA expression in bone marrow samples from 72 newly diagnosed AML patients,50 CR patients,8 relapse patients and 36 healthy donors by RT-PCR. Total RNA was isolated using TRIzol was applied to extract total RNA. Reverse transcription was performed using M-MLV reverse transcriptase cDNA Synthesis Kit. RT-PCR was used to analyze the expression levels of ZHX2 and NFYA.2. We quantitated ZHX2 mRNA expression among newly diagnosed AML patients, those reached CR after chemotherapy and when they relapsed. We further quantitated ZHX2 mRNA expression in different typing of AML patients of newly diagnosed. The diagnosis and classification of the patients were based on the revised French-American-British (FAB) classification. We also analyzed ZHX2 expression in newly-diagnosed AML patients with FLT3-ITD mutations and those with wild-type FLT3-ITD.3. Lentivirus-ZHX2 (LV-ZHX2) and lentivirus-ZHX2-RNAi (LV-ZHX2-RNAi) were purchased, as well as empty lentivirus. To analyze the ZHX2 level in AML cell lines, ZHX2 mRNA and protein expressions were examined in three AML cell lines (Kasumi-1, U937 and THP-1), a blast crisis CML cell line K562. AML cells were stably transfected with LV-ZHX2 and LV-ZHX2-RNAi, and incubated with puromycin, as well as empty lentivirus. We performed RT-PCR and Western blotting analyses to analyze the mRNA and protein expression levels of ZHX2, respectively.4. Cell viability was measured using the Cell Counting Kit-8 (CCK-8) was used to measure AML cell proliferation.5. Cells were treated with serial dilutions of cytarabine (Ara-C), Adriamycin (ADM) or Idarubicin (IDA) for 72h. Cell viability was assayed using the CCK-8, according to the manufacturer’s instructions.6. After treatment with IDA, ADM or Ara-c for 48 h, AML cells were harvested. Apoptosis was detected using an Annexin V Apoptosis Detection Kit APC, according to the manufacturer’s instructions. And fluorescence of cells was collected by Flow Cytometer to determine the percentage of apoptotic cells.7. Transfected AML cells were lysed in radio immunoprecipitation assay. The protein expression levels of Notch 1, NF-YA and DLL4 were detected in the AML cells by western blot.Results:1. The results showed that the expression of ZHX2 was low expression in newly diagnosed non-M3 AML patients and comparing to both CR patients and normal controls. Moreover, ZHX2 levels were significantly decreased in relapse patients compare with CR patients and healthy controls. There is no significantly different between CR patients and healthy controls. NF-YA mRNA expression level have no changed between in AML patients and healthy controls.2. The result showed that compare to the normal controls, ZHX2 is expressed at low levels in M2, M4 and M5 patients, especially in M5 patients. The data of tests suggested that the difference between without wild-type FLT3-ITD and the FLT3-mutated group was significant. The ZHX2 levels in CR patients were higher than those before any chemotherapy, but the results were not statistically significant and the ZHX2 levels were significantly increased when they relapse.3. The expression levels of ZHX2 were detected by RT-PCR and western blot analyses to verify AML cells were stably transfected with LV-ZHX2 and LV-ZHX2-RNAi.4. ZHX2 overexpression group inhibited proliferation by CCK-8 assays. Consistent with this result, the RNAi-mediated knockdown of ZHX2 in AML cell significantly had a proliferative advantage, compared to the empty lentivirus control.5. Compared with controls, knockdown of ZHX2 significantly decreased survival of THP1 cells exposed to different concentrations of ADM, Ara-c or IDA,. And transfection with the ZHX2 dramatically inhibited cell growth compared to transfection with the positive control.6. After labeled with Annexin V/PI, AML cells were tested by flow cytometry. These dates showed that apoptotic cells in ZHX2-transfected AML cells increased compared with those among control groups and apoptotic cells in RNAi-mediated knockdown of ZHX2 AML cells decreased compared with those among control groups.7. Western blot analysis showed that upregulation of ZHX2 resulted in the inactivation of the activated-Notchl with decreases in its ligand DLL4. In addition, knowdown of ZHX2 could also get roughly the same prediction.Conclution:1. The mRNA expression of ZHX2 was down-regulated in newly diagnosed and relapse non-M3 AML patients. These results indicated that ZHX2 expression may act as a prognostic factor associated with outcome of AML patients.2. Overexpression of ZHX2 in AML cell lines lead to suppress the cell proliferation of AML cell lines, increased sensitivity to chemotherapy drugs and inhibited the apoptosis of AML cell.3. ZHX2 is responsible for the notch pathway in AML cell lines.