SOX11 Inhibits Tumor Proliferation And Promotes Cell Adhesion Mediated-drug Resistance Via A CD43 Dependent Manner In Mantle Cell Lymphoma

Background:Mantle cell lymphoma(MCL)is a type of aggressive B-cell non-Hodgkin lymphoma(NHL).Due to its insidious onset,late clinical staging at the time of diagnosis,poor chemotherapy effect and easy recurrence,the median survival time of patients is usually only 3-5 years.Due to the unique expression of SOX11 in MCL,it has been widely used in clinical diagnosis,but its prognostic significance and underlying mechanism in MCL is still controversial.On the other hand,although CD43 is often expressed positively in MCL and its important role in cell proliferation,apoptosis,adhesion,migration has been shown in other malignancies,the relationship between CD43 and onset/progression of MCL still remain unknown.Objective:The aim of this study is to analyze the relationship between the expression of SOX11 and CD43 and the clinicopathological parameters of MCL.To investigate the significance of SOX11 and CD43 in the prognosis of MCL patients.To explore the role of SOX11 and CD43 on MCL cell proliferation as well as CAM-DR and reveal the related molecular mechanisms.Methods:1.Immunohistochemical and Immunofluorescence techniques were used to detect the expression of SOX11 and CD43 in the paraffin-embedded tissue of 67 patients with MCL.The correlation between the expression levels of SOX11 and CD43,as well as their relationship with the clinicopathological characteristics and survival prognosis of patients were retrospectively analyzed.2.The effects of silencing or overexpressing SOX11 and CD43 on the proliferation of MCL cells were examined by CCK-8 experiments and Ed U proliferation experiments.The effects of SOX11 and CD43 on MCL cell apoptosis were detected by flow cytometry.3.Real-time PCR and Western Blot were used to detect changes in m RNA and protein levels of CD43 after silencing or overexpressing SOX11 in MCL cells.The influence of SOX11 on SPN promoter activity was evaluated using dual-luciferase reporter assay,and the effect of CD43 overexpression on cell proliferation in SOX11 silencing MCL cells was investigated by rescue experiments.4.The effect of silencing or overexpressing SOX11 and CD43 on the MAPK signal pathway was detected by Western Blot,and the effect of the P38 MAPK signal on cell proliferation was detected by using SB203580(P38 MAPK inhibitor).5.We constructed a co-culture model of MCL cells and bone marrow stromal cells M2-10B4 or fibronectin(FN).Real-time PCR and Western Blot were used to detect the expression of SOX11 and CD43 in this model.6.The effects of silencing or overexpressing SOX11 and CD43 on the adhesion ability of MCL cells in co-culture models were tested by cell adhesion experiments.The effects of silencing or overexpressing SOX11 and CD43 on the chemoresistance to doxorubicin of MCL cells in the co-culture model were tested by cytotoxicity assay.The effect of CD43 overexpression on cell adhesion and chemoresistance in SOX11 silencing MCL cells was investigated by rescue experiments.Results:1.Prognostic significance of SOX11 and CD43 in mantle cell lymphoma Positive IHC staining for CD43 and SOX11 were detected in 49(73.1%)and 54(80.6%)cases.The expression levels of CD43 and SOX11 were distinctly correlated due to conegativity(P=0.036,Chi-square test).Moreover,it was also noteworthy that the negative correlation between SOX11 and Ki67 LI existed only in CD43 expression was positive rather than negative cases.The clinicopathological analysis showed that the positive expression of SOX11 was significantly correlated with Ki-67 <30%(P=0.009),normal LDH levels(P=0.014)and bone marrow involvement(P=0.008).CD43 positive expression was associated with Ki-67<30%(P=0.002)and normal LDH levels(P=0.010).Compared with the others(including SOX11-/CD43-,SOX11+/CD43-,SOX11-/CD43+ cases),the Ki-67 index and LDH levels were lower in the SOX11 / CD43 double positive group,but the proportion of bone marrow involvement was higher(P=0.001).Univariate survival analysis showed that patients with Ki-67<30%(P=0.011),normal LDH levels(P=0.008),MIPI low-risk group(P=0.004),CD43 positive expression and SOX11 /CD43 coexpression had longer overall survival(OS),while there were no significant differences in OS between the groups of age,gender,Ann Arbor stage,white blood cell count,presence or absence of B symptoms,and bone marrow involvement(P>0.05).Multivariate survival analysis showed that only Ki-67?30% was an independent influencing factor of poor prognosis in patients with MCL.2.Mechanism research of SOX11 and CD43 on cell proliferation and cell adhesion mediated drug resistance of mantle cell lymphoma Based on the expression of SOX11,we selected two MCL cell lines in vitro experiments,namely Je Ko-1 cell line with high expression of SOX11 and the JVM-2 cell line with low expression of SOX11.Silencing SOX11 with si RNA in Je Ko-1 cells could increase the phosphorylation levels of P38 MAPK and promote cell proliferation significantly,while exogenous overexpression of SOX11 in JVM-2 cells would weaken phosphorylation levels of P38 MAPK and inhibit cell proliferation.Pretreatment of Je Ko-1 cells with SB203580(P38 MAPK inhibitor)could significantly attenuate the proliferation-promoting effect induced by silencing SOX11,which indicated that silencing SOX11 promoted the proliferation of MCL cells by stimulating the P38 MAPK pathway.Silencing or overexpressing CD43 in MCL cells could also promote or inhibit cell proliferation which mediated by the P38 MAPK pathway.In addition,silencing SOX11 caused a decrease of CD43 expression in Je Ko-1 cells,while overexpression of SOX11 in JVM-2 cells could increase CD43 expression.The dual-luciferase reporter assay confirmed that SOX11 regulated the expression of CD43 by binding to the promoter region and promoting its transcriptional activity.Rescue experiments further confirmed that overexpression of CD43 could reverse the P38 MAPK phosphorylation and the proliferation of Je Ko-1 cells caused by silencing SOX11.In summary,the SOX11/CD43/ P38 MAPK signal axis in MCL was involved in regulating cell proliferation.Finally,we constructed a co-culture model to simulate the bone marrow microenvironment in vivo,and preliminarily explored the role of the SOX11/CD43 signal axis in CAM-DR.Real-time PCR and Western Blot showed that the expressions of SOX11 and CD43 in MCL cells adhering to bone marrow stromal cells M2-10B4 and FN were significantly up-regulated at m RNA and protein levels compared to suspension-cultured cells.When SOX11 or CD43 silenced,the adhesion of MCL cells to M2-10B4 cells or FN was significantly reduced,thereby weakening the CAM-DR effect and enhancing the sensitivity of MCL cells to doxorubicin.Conversely,overexpression of SOX11 or CD43 would promote adhesion and enhance the resistance of MCL cells to doxorubicin.The rescue experiment further confirmed that overexpression of CD43 can significantly reverse the decrease in adhesion and enhanced drug sensitivity caused by silencing SOX11.Therefore,we could consider that the SOX11/CD43 signal axis still exist in the bone marrow microenvironment of MCL and induce chemotherapy resistance by promoting MCL cells to adhere to bone marrow stromal cells or extracellular matrix.Conclusion:1.SOX11 and CD43 are positively expressed in most MCL cases,and their expression is significantly correlated.Patients with coexpression of SOX11/CD43 may be a MCL subtype with better prognosis which is related to lower Ki-67 index.2.SOX11 inhibits the proliferation of MCL cells and promotes the formation of CAM-DR by inhibition of the P38 MAPK pathway in a CD43 dependent manner,thereby providing new ideas for understanding the mechanism of action of SOX11 in MCL and reversing CAM-DR.