The Research On The Mechanisms Of Micro RNA150 Regulates B Lymphocytes In Autoimmune Hemolytic Anemia/Evans Syndrome Patients

Autoimmune hemolytic anemia is a kind of autoimmune disease characterized with abnormal activation of B lymphocytes and massive autoantibodies production.Autoantibodies produced by B cells can combine and destruct red blood cells with or without complement system mediation.Evans syndrome is a rare disease occurred when autoantibodies combine and destruct RBC and platelets simultaneously or sequentially.Although the specific mechanisms still are unclear,abnormal activation of B lymphocytes play an important role in the pathogenesis of the disease.Micro RNAs are 18²2nt nucleotide long small non-coding RNA molecules which can regulate gene expression post-transcriptionally by the combining of 3'untranslated region?UTR?of messenger RNA and repressing the whole process of translation.The translation process of one third mRNAs can be modulated by miRNAs.An increasingly great number of evident indicate that miRNAs play an important role in regulating immune cells develop,differentiation and is critical for the maintenance of immune balance.Previous studies had shown that the percentage of CD5⁺B lymphocytes cells?B1 cells?increased in AIHA/ES patients.The levels of CD80 and CD86 on CD5⁺B cells surface in newly diagnosed group were obviously higher than that of remission group and healthy controls?HCs?.The percentage of CD5⁺B lymphocytes which secrete IL-10 in newly diagnosed group was obviously higher than that of remission group and HCs.The level of miR-150 in CD19⁺B lymphocytes of AIHA/ES patients was lower and the expression level of c-Myb was higher than that of HCs.Both the expression level of miR-150 and c-Myb correlated with some clinical and immune features which can reflect the severity of disease.Based on previous studies,this research regulates the expression level of miR-150 in CD19⁺B lymphocytes of AIHA/ES patients and HCs by constructing plasmid expression vector and cell transfection.The percentage of CD5⁺B lymphocytes,the expression level of CD80,CD86 on B cells surface and the percentage of CD5⁺Bcells which secrete IL-10 in newly diagnosed group,remission group and healthy controls before and after transfection were detected with flow cytometry.The mechanisms which miR-150regulates B lymphocytes in AIHA/ES were explored.The level of miR-150 and c-Myb after transfection were detected and analyzed by qRT-PCR.The fact that miR-150 targets c-Myb in B cells of AIHA/ES patients were further validated.Finally,B lymphocytes'RNA was exacted by TRIzol reagent in AIHA/ES patients,HCs and CLL controls.The miRNA expression profile of AIHA/ES were screened and analyzed by BGISEQ-500 high throughput sequencing.The significantly differential expressed miRNA in AIHA/ES patients compared with healthy controls and CLL controls were selected out for future studies.Part 1 miR-150 regulates CD19⁺B lymphocytes'activity and secretive function inAIHA/Evans syndrome patientsObjective:To explore the mechanisms of miR-150 regulates CD19⁺B cells activity and secretive function in AIHA/Evans syndrome patientsMethods:8 hemolytic episode patients,7 remission patients and 5 HCs were enrolled in this study.Peripheral blood mononuclear cells?PBMNCs?were separated and CD19⁺B lymphocytes were sorted with magnetic activated cell sorting.Cells were cultured for 72h at 37?with an atmosphere of 5%CO₂ after transfected with hsa-miR-150-5p-inhibition plasmids and blank plasmids by Lipofectamine 3000reagents.The percentage of CD5⁺B lymphocytes,the level of CD80,CD86 on B lymphocytes and the percentage of CD5⁺B lymphocytes which secrete IL-10 in AIHA/Evans patients and HCs before and after transfection were detected with flow cytometry.Results:1 The percentage of CD5⁺CD19⁺/CD19⁺in hemolytic episode group?17.53±10.73%?was higher than that of remission group?6.15±2.30%,P<0.05?and HCs?7.03±2.14%,P>0.05?.There were no statistical differences between latter two groups.2 The level of CD80 on CD5⁺CD19⁺B in hemolytic episode group?29.11±10.97%?was higher than that of remission group?11.34±5.89%,P<0.01?and HCs?9.45±9.08%,P<0.05?.There were no statistical differences between latter two groups.The expression of CD86 on CD5⁺CD19⁺B in hemolytic episode group?32.23±10.64%?was higher than that of remission group?12.3±5.94%,P<0.01?and HCs?13.32±7.51%,P<0.01?.There were no statistical differences between latter two groups.There were no statistical differences of the expression of CD80 on CD5^-CD19⁺B among three groups?17.92±8.95%,21.02±10.73%,19.54±6.50%?.There were no statistical differences of the expression of CD86 on CD5^-CD 19⁺B among three groups?21.34±4.24%,15.18±7.40%,22.12±7.86%?.3 The expression level of IL-10 on CD5⁺CD19⁺B in hemolytic episode group?21.56±3.42%?was higher than that of remission group?8.19±3.66%,P<0.01?and HCs?7.54±3.67%,P<0.01?.There were no statistical differences between latter two groups.There were no statistical differences of the expression level of IL-10 on CD5^-CD19⁺B among three groups?3.19±1.06%,2.64±1.10%,3.62±0.79%?.4 After the transfection with hsa-miR-150-5p inhibition plasmids,percentage of CD5⁺CD19⁺/CD19⁺in hemolytic episode group?22.21±5.70%?was higher than that of before transfection?17.53±10.73%?without statistical difference.The percentage of CD5⁺CD19⁺/CD19⁺in remission group and HCs?11.66±1.62%,13.56±1.27%?was higher than that of before transfection?6.15±2.30%,7.03±2.14%?respectively?P<0.01?.After the transfection,the percentage of CD5⁺CD19⁺/CD19⁺in hemolytic episode group?22.21±5.70%?was higher than that of remission group?11.66±1.62%,P<0.01?and HCs?13.56±1.27%,P<0.05?.There were no statistical differences between latter two groups.5 There were no statistical different of the expression level of CD80 on CD5⁺CD19⁺B lymphocyte after the transfection with hsa-miR-150-5p inhibition plasmids compared with that before transfection.The expression of CD80 on CD5⁺CD19⁺B in hemolytic episode group?34.21±8.69%?was higher than that of remission group?10.64±3.05%,P<0.01?and HCs?11.73±2.45%,P<0.01?.There were no statistical differences between latter two groups.There were no statistical difference of the level of CD86 on CD5⁺CD19⁺B lymphocytes after the transfection with hsa-miR-150-5p inhibition plasmids compared with that before transfection.The expression of CD86 on CD5⁺CD 19⁺B in hemolytic episode group?27.17±8.72%?was higher than that of remission group?13.11±4.19%,P<0.01?.There was no statistical difference of the expression of CD86 on CD5⁺CD19⁺B lymphocyte between the hemolytic group and HCs.After transfection with inhibition plasmids,the differences of the expression of CD80,CD86 on CD5^-CD19⁺B among three groups couldn't be seen significantly compared with that after transfection.6 After transfection with hsa-miR-150-5p inhibition plasmids,the expression of IL-10 on CD5⁺CD19⁺B in hemolytic episode group,remission group and HCs?27.29±5.13%,21.01±4.50%,19.05±3.54%?was higher than that of before transfection?21.56±3.42%,8.19±3.66%,7.54±3.67%?and there were statistical differences among three groups?P<0.05?.The expression level of IL-10 on CD5⁺CD19⁺B in hemolytic group was higher than that of remission group?21.01±4.50%,P<0.05?and HCs?19.05±3.54%,P<0.05?after transfection.There were no obvious differences between latter two groups after transfection.There were no statistical differences of the expression of IL-10 on CD5^-CD 19⁺B compared with that before transfection among three groups.7 After the transfection with blank plasmids,the differences of percentage of CD5⁺B lymphocytes,expression of CD80,CD86 on B lymphocytes and percentage of CD5⁺Blymphocytes which secrete IL-10 couldn't be seen obviously compared with that before transfection in AIHA/Evans patients and HCs.Conclusions:The decreased expression of miR-150 in CD19⁺B lymphocytes of AIHA/Evans syndrome patients results in the increased percentage and the higher level of IL-10 in CD5⁺B lymphocytes.Part 2 c-Myb was the target of miR-150 in B lymphocytes of AIHA/Evanssyndrome patientsObjective:To verify that miR-150 targets c-Myb in B lymphocytes of AIHA/Evans syndrome patientsMethods:10 hemolytic episode patients,13 remission patients and 9 heathy controls?HCs?were enrolled in this study.Peripheral blood mononuclear cells?PBMNCs?were separated and CD19⁺B lymphocytes were sorted with magnetic activated cell sorting.Cells were cultured for 72h at 37?with an atmosphere of 5%CO₂ after transfected with hsa-miR-150 and hsa-miR-150-5p-inhibition plasmids by Lipofectamine 3000 reagents.Total RNA was extract with TRIzol reagent.The expression level of miR-150 and c-Myb in hemolytic group,remission group and HCs after transfection were detected and analyzed with qRT-PCR.Results:1 After transfected with hsa-miR-150 plasmids,the expression of miR-150 in CD19⁺B lymphocytes in hemolytic episode groups?0.64±0.21?was higher than that of before transfection?0.36±0.49?and the expression of miR-150 in B lymphocytes in remission groups?1.63±0.70?was higher than that of before transfection?0.76±0.73?without differences?P>0.05?.The expression of miR-150 in CD19⁺B lymphocytes in HCs?2.67±1.55?was obviously higher than that of before transfection?1.24±0.73,P<0.05?.The expression of miR-150 in CD19⁺B lymphocytes in hemolytic episode groups?0.64±0.21?was lower than that of remission group?1.63±0.70,P<0.05?and HCs?2.67±1.55,P<0.01?.There were no statistical differences between the latter two groups.2 After transfected with hsa-miR-150-5p inhibition plasmids,the expression level of miR-150 in CD19⁺B lymphocytes in hemolytic episode group?0.10±0.04?was lower than that of before transfection?0.36±0.49,P<0.05?;the expression of miR-150 in B lymphocytes in remission group?0.40±0.24?was lower than that of before transfection?0.76±0.73?without statistical differences?P>0.05?;the expression of miR-150 in CD19⁺B lymphocytes in HCs?0.50±0.16?was lower than that of before transfection?1.24±0.73,P<0.05?.The expression of miR-150 in CD19⁺B lymphocytes in hemolytic episode groups?0.10±0.04?was lower than that HCs?0.50±0.16,P<0.05?after transfection.There was no statistical difference between the remission group and HCs.3 After the transfection with hsa-miR-150 plasmids,the expression level of c-Myb in CD19⁺B lymphocytes was obviously lower than that of before transfection in hemolytic episode group?1.69±0.47,17.65±14.91,P<0.01?;remission group?0.54±0.83,9.23±8.22,P<0.01?and HCs?0.49±0.44,3.93±6.37,P<0.05?.The differences of c-Myb in CD19⁺B lymphocytes among three groups couldn't be seen.4 After the transfection of hsa-miR-150-5p inhibition plasmids,the expression level of c-Myb in CD19⁺B lymphocytes in hemolytic episode groups?12.30±2.49?was lower than that of before transfection?17.65±14.91?without statistical differences;the expression of c-Myb in CD19⁺B lymphocytes in remission groups?10.52±2.81?was higher than that of before transfection?9.23±8.22?without statistical differences.The expression of c-Myb in CD19⁺B lymphocytes in HCs?7.52±1.16?was higher than that of before transfection?3.93±6.37,P<0.05?.The expression of c-Myb in CD19⁺B lymphocytes in hemolytic episode groups?12.3±2.49?was higher than that of HCs?7.52±1.16,P<0.05?.There were no statistical differences of the level of c-Myb in CD19⁺B lymphocytes between the hemolytic group and remission group,remission group and HCs respectively.5 After cell transfection,the expression of CD19⁺B lymphocytes c-Myb were negatively correlated with the expression level of miR-150 in hemolytic episode group,remission group and HCs respectively?r=-0.8788,P<0.01;r=-0.6206,P=0.0136;r=-0.6606,P=0.0194?.Conclusions:miR-150 targets c-Myb in CD19⁺B lymphocytes and the decreased miR-150 attributes to the increased c-Myb in CD19⁺B lymphocytes in AIHA/Evans patients which may implicate the pathogenesis of the disease.Part 3 Screening and analysis of B lymphocytes'miRNA expression profile inAutoimmune hemolytic anemia/Evans syndrome patientsObjective:To analysis the expression profile of CD19⁺B lymphocytes micro RNA in AIHA/Evans syndrome patients.Methods:4 AIHA/ES newly diagnosed hemolytic episode patients,4 HCs and 4 CLL patients were enrolled in this study.PBMNCs were separated and CD19⁺B lymphocytes were sorted and purified with magnetic activated cell sorting for each sample.Total RNA was extracted by TRIzol reagent,and mixed equal proportionally as AIHA/ES group,HCs and CLL group.The expression profile of CD19⁺B lymphocytes'miRNA in AIHA/Evans syndrome patients,HCs and CLL patients were screened and analyzed after Small RNAs library construction and BGISEQ-500 high-throughput sequencing.Results:Compared with HCs,there were 178 miRNA up-regulated and 143 miRNA down-regulated expressed in AIHA/Evans syndrome groups.False discovery rate?FDR??0.001.Compared with CLL controls,there were 148 miRNA up-regulated and 186 miRNA down-regulated expressed in AIHA/Evans syndrome group.False discovery rate?FDR??0.001.Conclusions:The expression profile of CD19⁺B lymphocytes'micro RNA in AIHA/Evans syndrome patients was quite different from the expression profile of CD19⁺B lymphocytes'micro RNA in HCs and CLL respectively.These results provide scientific basis for future studies.