The Study Of The Clinical Significance And Mechanism Of Expression Of MiR-181/miR-195 In Diffuse Large B-cell Lymphoma

Diffuse large B-cell lymphoma(DLBCL) is a malignant lymphoma that is highly heterogeneous.Despite greater understanding of the DLBCL pathological subtypes and effectiveness of rituximab-based chemoimmunotherapy,35%–40% of patients show diminished treatment efficacy with rapid emergence of drug resistance.Therefore,there is an urgent need for in-depth understanding of DLBCL pathogenesis and the mechanisms that lead to drug resistance in order to improve survival rates.Micro RNAs(mi RNAs) are involved in tumorigenesis of many malignant tumors including DLBCL.They regulate cell differentiation,proliferation,apoptosis and other basic cellular processes and play an important role in tumor diagnosis,treatment and prognosis.In our previous study,we speculate that mi R-181a/b and mi R-195 may play an important role in the progression of diffuse large B-cell lymphoma.In this study,we investigated the clinical significance and mechanism of expression of mi R-181/mi R-195 in diffuse large B-cell lymphoma.PartⅠMicro RNA-181 a inhibits activated B-cell like diffuse large B-cell lymphoma progression by repressing CARD11Background: Activated B-cell(ABC)-like DLBCL related to the NF-κB pathway because aberrant activation related to the pathway are closely associated with cell survival and resistance of DLBCL cells to immunochemotherapy.Preliminary study demonstrated that mi R-181 a levels were lower in activated B-cell(ABC)-like DLBCL cells than in germinal center B-cell(GCB)-like DLBCL cells.Overexpression ofmi R181 a regulated the NF-κB pathway by repressing CARD11 and increases the therapeutic effects of immune-chemotherapy.Methods: Real-time PCR were performed to determine the mi R-181 a expression in ABC-like DLBCL.Bioinformatics method screen antiapoptotic gene regulated by mi R-181 a that expressed in ABC-like DLBCL.We builded dual luciferase reporter gene carrier and plasmid with the CARD11 3’UTR region,co-transfected of mi R-181a、anti-mi R-181 a inhibitor、control mimic in ABC-DLBCL cell.Luciferase reporter assay identify the target molecule of mi R-181 a.Western Blot detect target gene CARD11 expression before and after overexpressing mi R-181 a.Streaming annexin/PI double staining analyse of cell cycle and apoptosis by flow cytometry.To further confirm if mi R-181 a inhibits ABC-DLBCL tumorigenesis in vivo,we used the SCID mice xenograft model.Results: Mi R-181 a levels decreased in ABC-like DLBCL.Luciferase reporter assay and Western Blot show that mi R-181 a suppressed CARD11 obviously.Streaming annexin/PI double staining by flow cytometry display that the early and late apoptosis increased in mi R-181 a overexpressing cells than cells co-transfected of mi R-181 a with CARD11 and transfection control sequences.Tumor formation in nude mice show that injecting cells with mi R-181 a vector decreased tumor growth than injecting the control cells and injecting cells with mi R-181 a and CARD11 vector.Overexpression of mi R-181 a inhibits ABC-like DLBCL tumorigenesis by repressing CARD11.Conclusion: Overexpression of mi R181 a inhibits ABC-like DLBCL tumorigenesis by repressing CARD11.PartⅡ Expression and function of mi R-181 b in diffuse large B cell lymphomaBackground: Preliminary study demonstrated that the expression of mi R-181 b is closely related to invasiveness in malignant tumor.We speculate that the expression of mi R-181 b might relate with poor prognosis in malignant tumor.Method: The lymphoid tissues of 124 patients with diffuse large B cell lymphoma who were examined and treated from March 2010 to March 2012 were used as the observation group.The normal lymphoid tissues of another 64 patients with non lymphatic cancer were selected as the control group.The expression levels of mi R-181 b in two groups of samples were detected,and the expression levels of mi R-181 b in lymphoid tissues of lymphoma patients with different clinical features were compared.The survival of lymphoma patients with different levels of mi R-181 b expression was compared.Results: The relative expression of mi R-181 b in lymph tissues of DLBCL patients was significantly higher than that in normal lymphoid tissues(P<0.05).The higher the Ann Arbor staging was,the higher the relative expression of mi R-181 b was in lymph tissues(P<0.05),and the higher the IPI score was,the higher the relative expression of mi R-181 b was in lymph tissues(P<0.05).The 5-year survival rate of the patients with mi R-181 b low expression was significantly higher than that of the high expression group(P<0.05).Conclusion: There are differences in the expression of mi R-181 b in lymphoid tissues of DLBCL patients with different clinical stages and prognosis.It may become an effective indicator for the diagnosis and prognosis of DLBCL.PartⅢ Overexpressed mi R-195 attenuated immune escape of diffuse large B-cell lymphoma by targeting PD-L1Background: Diffuse large B-cell lymphoma(DLBCL)seriously threatens patients life with the morbidity increases at a high rate.Immune response disorder is the potential factor that induces DLBCL,while the potential mechanism still not fully understand.Methods: Real-time PCR were performed to determine expression of PD-L1 m RNA and mi R-195.Western blot were performed to determine PD-L1 protein levels.Flow cytometry was employed to detect the expression of PD-1 and the ratio of PD-1+T cells from peripheral blood.Enzyme-linked immune sorbent assay(ELISA)was used to determine the cytokines secretion.The mi R-195 mimic and mi R-195 inhibitor were transfected into cells using Lipofectamine 2000.Luciferase activities were measured by Luciferase reporter assay.Results: Mi R-195 level was down-regulated,while PD-L1 m RNA level was up-regulated in DLBCL tissues,and the rate of PD-1+T cells was increased in T cells of peripheral blood in DLBCL.The expression of mi R-195 was negatively correlated with PD-L1 in DLBCL patients.Cells transfected with mi R-195 mimic significantly promoted the expression of mi R-195,nevertheless,it decreased the expression of PD-L1.Overexpressed mi R-195 suppressed the expression of PD-L1.Moreover,mi R-195 overexpression significantly promoted the secretion of IFN-γ and TNF-α,but decreased IL-10 and PD-1+T cells rate in the co-culture model of T cells and OCI-Ly-10 cells.HEK293T cells transfected with mi R-195 mimic significantly decreased the luciferase activity in WT,and also inhibited the expression of PD-L1.HEK293 T cells transfected with mi R-195 inhibitor significantly promoted luciferase activity in WT,and accelerated the expression of PDL1.Mi R-195 targets PDL1 to promoted the secretion of IFN-γ,TNF-α,but decreased IL-10 and the rate of PD-1+T cells in the co-culture model of T cells and OCI-Ly-10 cells..Conclusion: Mi R-195 and PD-L1 might involved in the pathogenic mechanism of DLBCL.PD-L1 was the down-stream gene of mi R-195.Mi R-195 mediated the function of T cells.Mi R-195 targets PD-L1 to regulate its expression.mi R-195 targeting PD-L1 was mediated to the T cell function in response to DLBCL.In a word,mi R-195 regulated immune response of DLBCL through targeting PD-L1.