Objective: To investigate whether miRNA-30a is involved in the pathogenesis of ITP by affecting the differentiation of Th17 cells, and to explore its possible mechanism of miRNA-30a involved in the pathogenesis of ITP through the verification of the target gene SOCS3 for the prediction of miRNA-30a.
Methods: Firstly, a chronic ITP mouse model was established. The expression of miRNA-30a and RORγt in the spleen mononuclear cells were detected and their correlation were analyzed. Secondly, the luciferase vector containing 3'UTR of the target gene and green fluorescent vector containing miRNA were constructed. Luciferase fluorescence detection, real-time fluorescent quantitative PCR (qPCR) and Western blot were used to verify whether SOCS3 is the target gene of miRNA-30a.
Results: The platelet count of mice in experimental group decreased to below 20% of normal ones after 48 hours of injection of anti-mouse platelet serum (APS), which was maintained for 14 days at least; the expression of miRNA-30a and RORγt in the spleen mononuclear cells in experimental group were higher than those in the control group(P＜0.05), moreover, there was a positive correlation between them (r=0.54); the activity of luciferase in PMDH-GFP-miRNA-30a and pMIR-report-UTR was significantly lower than that in PMDH-GFP empty plasmid and pMIR-report-UTR(P＜0.05); The expression of SOCS3 at mRNA and protein level was not different from that in the control group.
Conclusion: Chronic ITP mouse model has been established successfully; miRNA-30a expression in spleen mononuclear cells of ITP mouse increase, and positively correlated with the expression of RORγt, which contribute to the pathogenesis of ITP by affecting the differentiation of Th17 cells; SOCS3 is able to bind to the target site of miRNA-30a, but might not be its functional target gene.
题目: miRNA-30a通过介导Th17细胞分化影响免疫性血小板减少性紫癜发病的初步探讨.
目的: 探讨miRNA-30a是否通过影响Th17细胞分化参与ITP发病；通过对miRNA-30a预测靶基因SOCS3的验证，进一步探讨miRNA-30a参与ITP发病的可能机制.
方法: 建立慢性ITP小鼠模型，检测其脾脏单个核细胞中miRNA-30a、 RoRγt的表达并分析两者相关性；构建含靶基因mRNA 3′UTR的荧光素酶表达载体及含有miRNA的绿色荧光载体，通过Luciferase荧光检测、实时荧光定量PCR（qPCR）、Western blot验证SOCS3是否为miRNA-30a的靶基因.
结果: 实验组小鼠血小板计数在注射抗小鼠血小板血清（APS）48 h后下降至正常的20%，且至少可维持14 d。实验组小鼠脾脏单个核细胞中miRNA-30a及RoRγt表达较对照组升高（P＜0.05），且两者存在正性相关（r=0.54）。pMDH-GFP-miRNA-30a与pMIR-report-UTR实验组的荧光素酶的活性明显低于pMDH-GFP空质粒与pMIR-report-UTR的对照组（P＜0.05）。转染miRNA-30a质粒的EL4细胞中SOCS3在mRNA及蛋白水平与对照组均无差异.
结论: miRNA-30a在ITP小鼠脾脏单个核细胞中表达升高，且与RORγt表达呈正相关性，它可能通过影响Th17细胞分化参与ITP发病；SOCS3能与miRNA-30a靶位点结合，却不是其功能靶基因.