Study On The Role And Mechanism Of Myeloid Differentiation Factor MyD88 In The Plasma Cell Diseases

Part I The clinical features of patients with lymphoplasmacytic diseases harboring MyD88 L265 P mutation Objective To establish the method of ARMS PCR-Capillary Electrophoresis(ARMS PCR-CE)and explore the role of ARMS PCR-CE in the detection of MyD88 L265 P mutation and analyze the clinical features of MyD88 L265 P mutated patients with lymphoplasmacytic diseases.Methods To analyze the distribution of MyD88 L265 P mutation in patients with lymphoplasmacytic diseases by using of ARMS PCR-CE.Results ARMS PCR-CE assay improved the positive detection rate of MyD88 L265 P mutation.There were 25(30.9%)MyD88 L265 P mutated patients in 81 patients.The mutation was frequently observed in 14 patients with WM(77.8,14/18),2 patients with lymphoplasmacytic lymphoma(66.7%,2/3),1 acute lymphocytic leukemia patient(50%,1/2),3 multiple myeloma patients(30%,3/10),1 patient with monoclonal gammopathy of undetermined significance(25%,1/4),3 patients with chronic lymphocytic leukemia(13%,3/23)and 1 lymphoma patient(4.8%,1/21).20(80%,20/25)patients were identified with IgM subtype.Compared with wild group of 56 cases,mutated patients were older(median age:67 years VS 55 years,P<0.001),with lower white blood cell count(median count:5.23×10~9/L VS 10.8×10~9/L,P=0.001),lower hemoglobin count(median count:85g/L VS119 g/L,P<0.001).Based on the median count of MyD88 L265 P mutation quantification(3%),all the 25 mutated patients were defined as high expression group(?3%)and low expression group(<3%).There were more male patients(P=0.030)in the high expression group.The patients in the high expression group had lower hemoglobin count(median count:69g/L VS 94g/L,P=0.044)and lower platelet count(median count:77×10~9/L VS244×10~9/L,P=0.001).Conclusion ARMS PCR-CE assay could increase the positive detection rate of MyD88 L265 P mutation.MyD88 L265 P mutation mainly presented in IgM subtype lymphoplasmacytic diseases,especially for Waldenstrom's macroglobulinemia.Compared with the wild group,patients with MyD88 L265 P mutation were older and had lower white blood cell and lower hemoglobin count.However,the significance of the prognosis of patients with MyD88 L265 P mutation needed further studies.Part II The role and mechanism of MyD88 gene in multiple myeloma cell growthObjective To study the effect of MyD88 on multiple myeloma cell growth and explore the role and mechanism of MyD88 in multiple myeloma cells.Methods We constructed the lentiviral vector which highly expressed MyD88.Virus was packaged by this lentiviral vector to establish the stable U266 and LP1 cell lines highly expressing MyD88.MyD88-targeting sh RNA was transiently transfected into U266 cell line with liposome to interfere the expression of MyD88.With the over-expression and down-expression of MyD88,we used CCK8 kit to detect myeloma cell proliferation,flow cytometry to detect myeloma cell's apoptosis,Real-Time PCR to detect the expressions of IL-6,MCP-1,TGF-?,TNF-?,VEGF,IFN-?,IL-10 cytokines,and c DNA Microarray to detect the change of multiple myeloma's gene expression profile with the over-expression of MyD88 gene.Results We detected MyD88 expression of six myeloma cell lines(RPMI8226?L363?KMS11?LP1?mm1s and U266)in our center,and all of them could be detected MyD88 expression.We then chose LP1 cell line as the low MyD88 expression cells and U266 cell line as the high MyD88 expression cells in the further study.Firstly,we constructed MyD88 stable cell lines: U266-MyD88 and LP1-MyD88.The OD450 values of U266-MyD88 cells when cultured for 24 h(P<0.001),48 h(P=0.043)and 72 h(P=0.024)were significantly higher than those of U266-Venus,and the OD450 values of LP1-MyD88 cells when cultured for 24 h(P<0.001),48 h(P<0.001)and 72h(P=0.001)were also higher than those of LP1-Venus.Moreover,the OD450 values of the U266 cells interfered with MyD88-targeting sh RNA for 24 h(P=0.023),48 h(P<0.001),and 72 h(P<0.001)were less than those of the control group.Secondly,the early apoptosis(1.94% VS 1.14%,P=0.002)and total apoptosis(2.983% VS 2.317%,P=0.013)in MyD88 down-expression cells were higher than that of U266-Venus.Thirdly,IL-6,MCP-1,TGF-?,TNF-?,and VEGF were increased in U266-MyD88 cells,and TNF-? was about 4.9 times compared to the control.On the contrary,IL-6,MCP-1,TNF-?,VEGF,IFN-?,IL-10 were decreased in the U266 cells with down-expression of MyD88.IFN-? and IL-10 were 0.09 and 0.12 times to the control group,respectively.With c DNA microarray we found that there were 171 up-regulated genes and 91 down-regulated genes in U266-MyD88 cells compared to U266 cells,and pathway analysis suggested that most of the up-regulated genes participating the NF-k B signaling pathway.With 10 N Bortezomib treatment,we found that the inhibition of cell proliferation of U266-MyD88 cells in 48 h and 72 h were 51.6% and 69.6%,respectively,while the inhibition of cell proliferation of U266-Venus cells in 48 h and 72 h were 31.5% and 49.3%,respectively.With 20 N doses Bortezomib treatment,the early apoptosis(4.19% VS 2.61%,P=0.022),late apoptosis(0.815% VS 0.31%,P<0.001)and total apoptosis(5.005% VS 2.92%,P=0.006)of U266-MyD88 cells were all higher than U266-venus cells.Conclusion 1)MyD88 could play an important role in multiple myeloma cell growth.Interfering MyD88 could inhibit cell proliferation and promote the apoptosis of multiple myeloma cell lines.NF-k B pathway might be activated by over-expressed MyD88,which led to the high expression of IL6,MCP-1,TGF-?,TNF-? and VEGF,and then enhanced the proliferation of multiple myeloma cell lines.2)High expression of MyD88 further improved the sensitivity of Bortezomib treatment.Inhibition of NF-k B pathway by Bortezomib could promote the apoptosis of the stable cell line with high expression of MyD88.This study can help to further understand the mechanism of MM and provide theoretical and experimental basis for developing new targeted drugs.