Impact And Mechanism Of Overexpression Of ZNF521 On AML Cell Lines And Meta-analysis Of The Prognostic Value Of CD11b Expression For AML Patients

PART I Impact and mechanism of overexpression of ZNF521 on AML cell linesBcakground:Acute myeloid leukemia, also known as acute non-lymphocytic leukemia, is a highly malignant disease originated from hematopoietic stem or progenitor cells. In China, acute myeloid leukemia is the most common type of leukemias in adults, and the incidence increased with age, the progression is usually rapid, and the natural course of the disease is only a few months.The occurrence of leukemia is a multi-step process caused by a variety of genetic abnormalities and epigenetic changes, the molecular mechanism of which was so complicated that still unable to get clearly elaborated. There were a lot of studies indicating that the abnormal regulation of transcription factors,which made hematopoietic stem cells unable to differentiate into the functional mature blood cells, was the important machanism of leukemia.In recent years, zinc finger protein 521(ZNF521) had attached attention as an important transcription factor, for its important role in regulation of the maintenance and differention direction of hematopoietic stem cells. What’s more important, the high expression of ZNF521 was found in most of the AML patients. At the same time, the high expression of ZNF521 was significantly correlated with the specific genetic abnormality, especially MLL gene rearrangement. These studies indicated that ZNF521 may be involved with the occurrence of leukemia, but how ZNF521 play a role in the occurrence of leukemia and its mechanism is still not clear. Therefore, to deeply investigate the effect of ZNF521 on the proliferation and apoptosis of acute myeloid leukemia cells and clarify its molecular targets is of great importance for further clarifying the pathogenesis of AML and enrichingits treatment.Objectives:1.To construct the plasmid p CDH-CMV-puro-ZNF521 using molecular clone method. Establish gene overexpression stable cell lines including ZNF521-K562 and ZNF521- THP-1 by lenti viral infection system. At the same time, package lenti viral vector p CDHCMV-puro into lentiviral particles and establish NC-K562 and NC-THP-1 stable cell lines as controls.2. Detect the impact of ZNF521 overexpression on proliferation and apoptosis of AML cell lines K562 and THP-1 by CCK-8 assay and Annexin-V/PI.3. Through detecting ZNF521 downstream targets that were associated with cell proliferation and apoptosis, try to find the possible mechanism of the impact of ZNF521 overexpression on AML cell lines.Methods:1. Amplify p Tango-zeo-N4Flag-ZNF521 plasmid. ZNF521 cds sequence was searched by NCBI, confirming p Tango-zeo-N4Flag-ZNF521 expressed human protein ZNF521 isoform1. Using the vector NTI to design the primers targeting N4Flag-ZNF521 fragment for PCR amplification, and the 5 ’end and the 3’ end of the primers were added with Nhe I and Swa I restriction sites respectively. Then the amplification products were digested by Nhe I and SWAI restriction enzyme and connected into p CDH-puro lentiviral vector, and transformed into competent E.coli DH5α. The positive clones were selected to extract the plasmid. Thus we obtained the p CDH-puro-ZNF521 lentiviral plasmid. After identification by enzyme digestion, p CDH-puro-ZNF521 and the empty vector p CDH-puro were transfected into HEK293 T with ps PAX2 and p MD2.G cells, thus we obtained lentiviral particles containing ZNF521 and the controls.2. After the above cell lines were infected by corresponding lintivirals.we used puromycin at 2ug/ml to select the successfully infected cells, and observed cell activity 5 days later. Then we extracted RNA and protein of each cell lines, and used Real-Time PCR and Western Blot to detect the overexpression of m RNA and protein of ZNF521.3. Use CCK-8 Cell Proliferation Assay Kit and Annexin-V/PI Cell Apoptosis Assay Kit respectively to detect the impact of ZNF521 overexpression on proliferation and apoptosis of K562 and THP-1.4. Use Real-Time PCR and Western Blot to detect c-myc expression level in ZNF521-K562 and ZNF521-THP-1 and their corresponding control.Results:1. p CDH-puro-ZNF521 plasmid was constructed successfully, and ZNF521-THP-1, ZNF521-K562, Vcetor-K562 and Vector-THP-1 stable cell lines were successfully established。2. CCK-8 cell growth curve showed that both of ZNF521-K562 and ZNF521-THP-1 presented notably higher proliferation rate compared with their corresponding controls.3. Flow cytometry indicated that ZNF521 overexpression had no significant impact on cell apoptosis.4. The expression level of c-myc m RNA and protein was upregulated in ZNF521-THP-1and ZNF521-THP-1compared with their corresponding controls.Conclusion:ZNF521 overexpression could significantly promote cell proliferation of AML cell lines K562 and THP-1, but its role in cell apoptosis was not notable. The promotion effect of ZNf521 overexpression in cell proliferation was possibly induced by the activation of c-myc expression.PART Ⅱ Systemetic review of the prognositic value of CD11 b expression for Acute Myeloid Leukemia patientsObjectives:The aim of this meta-analysis is to evaluate the value of CD11 b expression level for prognosis of AML patients.Methods:We searched Pub Med, Embase, Cochrane Library, Chinese Bio Medical Literature Database(CBM) and Web of Science(from when these electronic databases were established to July,2015) to achieve studies that assessed the impact of CD11 b expression level on the prognosis of AML patients. We performed the literature selection based on the predefined search strategy, and then quality evaluation, data extraction and meta analysis were carried out. Disease-free survival(DFS),overall survival(OS) and complete remission rate(CRR) were selected as evaluating indicators. We used the software Revman5.3 to perform this meta-analysis to assess the prognostic value of CD11 b for AML patients comprehensively and reasonably.Results:A total of 13 studies involving 2619 subjects were included. Meta analysis showed that the complete remission rate of CD11 b positive AML patients was significantly lower than that of CD11 b negative patients(OR = 0.44; 95% 0.25-0.79, CI; P = 0.006), and the overall survival of CD11 b positive AML was also significantly shorter(HR = 0.66; 95% 0.55-0.80, OS; p; CI; CI; HR; 0.31-1.48; CRR; P = 0.32). While the difference of disease-free survival of these two groups was not apparent(HR = 0.67; 95% CI, 0.31-1.48; p = 0.32). The subgroup analysis results showed that cut-off value for CD11 b positivity, ethnicity, subtype, treatment and sample preparation method had no significant interaction on the prognostic role of CD11 b expression level on AML patients. The sensitivity analysis that only included NOS high score studies showed consistent result with the whole meta analysis.Conclusion:CD11b positivite expression could endow a poor prognosis for AML patients. The results of this meta-analysis also indicated that CD11 b holds promise to be a new prognostic biomarker for AML patients.