| Myelodysplastic syndromes（MDS）are a group of heterogeneous acquired clonal disorders.The basic pathological changes are dysplasia of clonal hematopoietic stem progenitor cells,which leads to low hematopoietic function and decreased bone marrow function,and may increase the risk of malignant transformation.The number of hematopoietic cells in bone marrow increased or was normal,but there were obvious morphological changes of abnormal development.There is a significant reduction in blood cells and a high risk of developing acute myeloid leukemia（AML）.This process was gradual and was mediated by abnormalities in the potentially lost signal transduction pathways of various cytokine tumor suppressor genes and by immune mechanisms.All-trans retinoic acid（ATRA）has been proved to be widely used in the treatment of hematological diseases,such as acute promyelocytic leukemia（APL）.However,due to its severe retinoic acid syndrome,drug resistance,hepatorenal toxicity and other problems,the clinical application of ATRA has been limited to some extent.Therefore,a series of novel retinoic acid derivatives were designed and synthesized with ATRA as a lead compound.Among these derivatives,4-amino-2-trifluoromethyl-phenyl retinate（ATPR）was confirmed by the group’s previous pharmacological experiments to induce the differentiation of multiple tumor cells and inhibit cell proliferation in vitro,but the specific molecular mechanism of ATPR on tumor cells remains to be further investigated.To further analysis about ATPR for induction of MDS,possible mechanisms of cell differentiation and the dependence of RNA helicase DDX23 in ATPR role differentiation process,In this study,the SKM-1 cell line was selected for the experiment.Through proteomics analysis of gene differential expression,in vitro experiments were conducted to observe the effect of ATPR on the expression of ATP-dependent RNA helicase DDX23 and the role of DDX23 in the process of ATPR induction of SKM-1 cell differentiation.The research content of this project can be divided into the following three parts:1. Effects of ATPR on SKM-1 cell differentiation and cycleSKM-1 cells were treated by ATPR at different concentrations(0,10-5,10-6,10-7,10-8,10-9 M)and at different time points（24,48,72h）.Then,the proliferation activity of SKM-1 cells was detected by CKK-8 method to determine the optimal conditions of action of ATPR on SKM-1 cells.Then,western blot was used to analyze the expression changes of differentiated proteins p27 and PU.1 under different conditions.And after ATPR(10-6 M)was treated to SKM-1 cells for 72h,the changes in the cell cycle were detected by flow cytometry.The results indicated that ATPR could increase the expression of differentiated proteins in SKM-1 cells in vitro,and the effect was significant at the concentration of 10-6 M and the duration of action was 72h.The results showed that ATPR could block SKM-1 cells in G1 phase.2. Proteomics analysis of the effects of ATPR on SKM-1 cells.After applying ATPR(10-6M)to the cells for 72 hours,the cells were collected,and the protein was compared in different samples through the comparison of MS spectra and then integrated into the corresponding proteins through the proteomics non-standard quantitative technology（Label-Free）.Finally analyzed the protein expression difference between ATPR effect and SKM-1 cells and verified.The results suggest that a total of212 up-regulated proteins with a difference greater than 1.2 times and 18down-regulated proteins with a difference less than 5/6 times were screened.The verified results are true,among which ATP-dependent RNA helicase DDX23 has the greatest effect when ATPR acts on SKM-1 cells.3. The role of DDX23 in the differentiation of SKM-1 cells induced by ATPRThe overexpressed plasmid GV146-DDX23 was transfected into SKM-1 cells,and ATPR(10-6 M)was used to stimulate the transfected SKM-1 cells.And then Western blot was used to detect the expression levels of DDX23,Notch1,P27,PU.1,PCNA,CDK4 and CDK2 in the cells.The morphological changes of cells were detected by Wright-Giemsa staining.The expression of mononuclear line differentiation antigen CD14 on the cell surface was detected by flow cytometry.The results showed that overexpression of DDX23 could further promote the decreased expression of Notch1,CDK2,CDK4 and PCNA in SKM-1 cells caused by ATPR,while the protein expression changes of PU.1 and p27 were reversed.At the same time,overexpression of DDX23can further enhance the morphological changes of SKM-1 cells induced by ATPR to mature cells and the expression of the mononuclear differentiation antigen CD14 on the surface of SKM-1 cells.To further confirm the role of DDX23,we used si RNA-DDX23 to reduce the expression of DDX23 in SKM-1 cells and then used Western blot to detect the expression of DDX23,P27,PU.1,PCNA,CDK4 and CDK2 proteins in the cells;Morphological changes of the cells were detected by Wright-Giemsa staining;The expression of CD14,a monocyte differentiation antigen,on the cell surface was detected by flow cytometry.The results showed that DDX23 silencing could reduce the expression of p27 and PU.1 in SKM-1 cells,while slightly increasing the expression of CDK2,CDK4 and PCNA.In addition,DDX23 silencing could antagonize the differentiation of SKM-1 cell morphology towards maturity induced by ATPR and down-regulate the expression of surface monocyte differentiation antigen CD14 in SKM-1 cells. |
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