| Mantle cell lymphoma（MCL）is a lymphoproliferative disorder disease lacking reliable therapies.New treatment approaches to target novel biological pathways are needed.4-Amino-2-Trifluoromethyl-Phenyl Retinate（ATPR）synthesized by our group has been previously proven to have higher solubility and superior differentiation effect compared with the conventional all-trans retinoic acid in acute myeloid leukemia.Previous study has proved that ATPR could induce differentiation and inhibit proliferation of acute promyelocytic leukemia.However,whether ATPR could induce differentiation from MCL cells to normal immune cells has never been investigated.In this study,we found that the proliferation of JEKO-1 cells was completely repressed and the differentiation was activated after ATPR treatment.We further found that the neural transcription factor SOX11 was highly expressed in MCL but downregulated by ATPR.After silencing SOX11 in vitro and vivo,the malignant proliferation and inhibited differentiation of JEKO-1 cells were reversed,while the over-expression of SOX11exacerbated the malignant phenotype of JEKO-1 cells.In addition,the E2F1 protein functions as a transcription factor that enhances cell proliferation by binding to the promoter regions of various genes,including those involved in cell cycle regulatory activities and DNA replication.Dysregulation of the Rb/E2F1 pathway promotes tumor development by deregulating the E2F family of transcription factors,resulting in uncontrolled cell cycle progression.It has been shown that during cell cycle progression,cyclin D activates cyclin-dependent kinases CDK4/6 to inactivate RB,allowing S-phase gene transcription of E2F1.the Cyclin D1/Rb/E2F1 axis was involved in MCL and regulated by ATPR.Conclusively,we found that ATPR promoted JEKO-1 cells differentiation via SOX11/Cyclin D1/Rb/E2F1.This study provides experimental evidence for the future induced differentiation therapy of MCL with ATPR.Objectives:This study aimes to explore the role of SOX11 in ATPR-induced differentiation of human mantle cell lymphoma cells and its mechanism.Methods:1.Preliminary study of the effect of ATPR on the differentiation of human mantle cell lymphoma cells in vitroIn order to explore the effect of human mantle cell lymphoma JEKO-1 cell line on differentiation after ATPR treatment,the Giemsa staining method was used to observe ATPR at different concentrations(10-5 M,10-6 M,10-7 M)and at different times（0,24,48,72 h）on cell morphological changes,followed by western blot and flow cytometry to detect ATPR at different concentrations(10-5 M,10-6 M,10-7 M)and at different times（0,24,48,72 h）on the surface differentiation antigens CD138and CD38.Finally,we screened out different concentrations(10-5 M,10-6 M,10-7 M,10-8 M,10-9 M))by CCK8.The inhibitory effect of ATPR on the proliferation of human mantle cell lymphoma cells2 In vitro experiments to explore the effect of ATPR on the expression of SOX11 in mantle cell lymphoma cellsCD34-positive cells from umbilical cord blood were identified by flow cytometry,and the protein expression of SOX11 in different cell lines was detected by western blot.A positive drug control experiment was set up,and western blot and immunofluorescence techniques were used to determine the distribution of SOX11 in cells.Then western blot was used to detect the effect of ATPR on SOX11 at different concentrations(10-5 M,10-6 M,10-7 M,10-8 M,10-9 M)and at different times（0,24,48,72 h）.Effects on protein expression and m RNA levels.3.Silencing or overexpressing SOX11 in ATPR-induced differentiation and cycle arrest of mantle cell lymphoma in vitro and in vivo experimentsTo investigate the role of SOX11 in ATPR-induced mantle cell lymphoma differentiation and cycle arrest,lentivirus silencing or overexpression of SOX11,western blot,q PCR and immunofluorescence techniques were used to verify the transfection effect.The proliferation of cells after silencing or overexpression of SOX11 was observed by immunofluorescence,the expression of Prb,Cyclin D3,Cyclin A2,and CDK4 proteins was detected by western blot,and the cell morphology was observed by Giemsa staining.Changes of CD138 and CD38 after ATPR treatment.Cell apoptosis was detected by flow cytometry.The mouse model of NSG xenograft tumor was constructed in vivo,the effect of SOX11 silencing on tumor growth in mice was observed,and the protein expression changes in mouse tumor tissues were detected by western blot and immunohistochemistry.4 In vitro experiments to further explore the role of ATPR through the SOX11/Cyclin D1/RB/E2F1 signaling pathwayWestern blot was used to detect the effect of ATPR on Cyclin D1 at different concentrations(10-5 M,10-6 M,10-7 M,10-8 M,10-9 M)and at different times（0,24,48,72 h）.The protein expression of Prb and E2F1 was affected by silencing and overexpression of SOX11,respectively,and the changes of Cyclin D1/RB/E2F1 pathway protein levels were observed.Results:1.The results of Giemsa staining showed that the effect of ATPR was 10-5 M for 72 hours on the nuclear contraction of JEKO-1 cells,and the best differentiation effect.Western blot and flow cytometry showed that the protein expression of CD138 and CD38 in JEKO-1 cells increased the most and the curve shifted to the right most obviously when the concentration of ATPR was 10-5M for 72 hours.and the results of CCK8 also showed that the best inhibitory concentration of ATPR was 10-5 M.2.Flow cytometry and western blot results showed that SOX11 was highly expressed in human mantle cell lymphoma nuclei,and ATPR could inhibit the expression of SOX11in a time-and dose-dependent manner.3.The experimental results showed that after silencing SOX11,the fluorescence expression of SOX11 was significantly weakened,the ratio of G0/G1 phase was reduced,the expressions of cell cycle-related proteins Prb,Cyclin D3,Cyclin A2,and CDK4 were reduced,while the expression of cell surface differentiation antigens CD138 and CD38proteins decreased.Increase.The results of Giemsa staining also showed that after silencing SOX11,the nuclei of JEKO-1 cells contracted into a kidney type,and the nucleocytoplasmic ratio decreased,indicating a high degree of cell differentiation.Flow cytometry results showed that the number of positive cells for cell surface differentiation antigens CD138 and CD38 increased,Flow cytometry results also showed that silencing SOX11 could induce apoptosis.The above experimental results were enhanced after ATPR treatment.Overexpression of SOX11 with lentivirus significantly enhanced the fluorescence expression,increased the ratio of G0/G1 phase,and increased the expression of cell cycle-related proteins Prb,Cyclin D3,Cyclin A2,and CDK4,while the expression of cell surface differentiation antigens CD138 and CD38 decreased.The results of Giemsa staining also showed that after overexpression of SOX11,cell differentiation was not obvious,and flow cytometry results showed that the number of positive cells for cell surface differentiation antigens CD138 and CD38 decreased,and flow cytometry results also showed that overexpression of SOX11 inhibited cell apoptosis.Death.However,the above experimental results were similiar to the normal group after co-treatment of overexpression of SOX11 and ATPR,suggesting that overexpression of SOX11 reversed the effect of ATPR-induced differentiation and cycle arrest in mantle cell lymphoma.4.The results showed that the protein expressions of Cyclin D1,Prb,and E2F1 were decreased after ATPR treatment.After silencing and overexpressing SOX11,the results showed that the protein expression levels of phosphorylated RB,E2F1,and Cyclin D1were decreased after ATPR treatment and silencing of USO1.Overexpression of SOX11reversed the inhibitory effect of ATPR.Conclusion:ATPR affects the Cyclin D1/RB/E2F1 pathway by inhibiting the expression of SOX11,and finally induces the differentiation and cycle arrest of human mantle cell lymphoma cells. |
PDF Full Text Request