| Background and Objectives:Acute myeloid leukemia(AML) is the most common type of leukemia that affects adults, with a prevalence of 3.8 cases per 10000 rising to 17.9 cases per 10000 adults aged 65 years and older. It is a heterogenous clonal disorder of hematopoietic stem or progenitor cell which lose the ability to differentiate normally and to respond to normal regulators of proliferation and apoptosis, leading to the inhibition of the normal hematopoiesis process and infiltration of different organs, causing anemia, bleeding, infection and other clinical manifestations and eventually death.Because its clinical heterogeneity, prognosis of different AML patients varies a lot. Although cytogenetic methods were currently widely used to predict the prognosis of AML, but this criteria still can not meet the clinical needs, so it is necessary to find new biomarkers with prognostic significance.Protein post-translational modification by small ubiquitin like modifier(SUMO) attracted widespread attention due to its simplicity and functionality. SUMO modification is a dynamic process, and SUMO specific proteinase 1(SENP1) is the strongest de-SUMOylation protease. Previous studies showed that SENP1 is a critical regulator of the development of red blood cells and also play important roels in T cells and B cells development, suggesting the key role of SENP1 in normal hematopoiesis. But expression levels, biological effects, mechanisms and prognostic significance of SENP1 in AML patients are currently unknown.In order to explore the above unanswered question,we conducted this study which includeds three parts. The first part consistes of detecting SENP1 expression level in AML patients by RT-PCR, immunohistochemistry and western blot, investigating the effects of SENP1 on proliferation and apoptosis of AML cells by building SENP1 knockdown stable cell lines, analyzing the prognostic role of SENP1 expression level by Kaplan- Meier analysis and observing the correlation between SENP1 and known prognostic biomarker CD56, CD11 b by correlation analysis. The second part of this study includes investigating the impact of SUMOylation of HOXA9 on the biological characterisitics of AML and the impact of SUMOylation of ZNF521 on the biological characteristics of AML, aming to explain the mechanisms of SENP1 knockdown effects. The third part investigates the prognostic significance of CD56 and CD11 b, which are both statisticaaly correlated with SENP1, to provide clues for explaining the prognostic effects of SENP1.Material and Methods:The entire study adopted methods includes sample experiments, cell experiments, animal studies, clinical data analysis. Smaple expreriments are based on previously collected bone marrow mononucleate cells from AML patients and normal bone marrow. Cell experiments in conducted by manipulating representative AML cell lines(such as THP-1, K562, U937). Animal experiments used AML NOD / SCID mice model constructed by transplantation. Clinical data analysis is mainly based on information available in public databases.1. Detected SENP1 protein and mRNA expression level in AML samples by RT-PCR, immunohistochemistry and western blot respectively.2. Observed the effect of SENP1 on AML cell proliferation and apoptosis by building SENP1 knockdown stable AML cell lines; Adopted RNA-sequencing method to find possible downstream genes of SENP1.3. Applied Kaplan-Meier analysis and correlation analysis to investigate the prognostic significance of SENP1 for AML pateints and investigated the correlation between SENP1 and other prognostic molecular reported(CD56, CD11b).4. Investigated the effects of SUMOylation of HOXA9 and ZNF521 on the biological characteristics of AML which can help to explain the effects of biological characteristics of SENP1 by co-immunoprecipitation, point mutation, stable transfected cell line construction.5. Validated prognostic significance of SENP1’s relevant molecules of CD56 and CD11 b, provided clues for explaining the prognostic role of SENP1.Result:1. SENP1 protein and m RNA was highly expressed in AML.2. Compared with control, the proliferation of AML cell lines reduced, the proportion of apoptosis increased in SENP1 knockdown stable AML cell lines.3. SENP1 overexpression is associated with poor prognosis of AML patients, and there is a significant correlation between SENP1 and known prognostic molecules CD56 and CD11 b.4. HOXA9 can be SUMOylated, and deficiency of SUMO modification(point mutation) enhanced the pro-proliferation ability of HOXA9. SUMOylation of HOXA9 is regulated by SENP1.5. ZNF521 can be SUMOylated, and deficiency of SUMO modification(point mutation) enhanced the pro-proliferation ability of ZNF521. SUMOylation of ZNF521 is regulated by SENP1.6. Prognostic value of CD56 and CD11 b which are significant correlated with SENP1 are validated by the method of meta-analysis.Conclusion:1. SENP1 is highly expressed in AML samples, with the ability to promote AML cell proliferation and inhibit apoptosis.2. SENP1’s effects on the biological characteristics of AML may be explained by regulating the SUMOylation status of HOXA9 and ZNF521.3. Significant correlated molecules of SENP1, CD56 and CD11 b are validated to be of prognostic value of AML which provided clues for explain the prognostic role of SENP1 in AML. |
