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Updated: Dec 23, 2025
Isolation and Th17 Differentiation of Naïve CD4 T Lymphocytes
Published on: September 26, 2013
[Pathogenesis of Immune Thrombocytopenic Purpura (ITP) by MiRNA-30a-Mediated Th17 Cell Differentiation]
Xiao-Fang Wu1, Jian-Qin Li2, Shao-Yan Hu1
1Department of Hematology, Children's Hospital Affiliated to Soochow University, Suzhou 215000, Jiangsu Province, China.
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.
Insights
MicroRNA-30a (miRNA-30a) levels increase in immune thrombocytopenia (ITP) and correlate with Th17 cell differentiation, contributing to ITP pathogenesis. SOCS3 binds miRNA-30a but may not be its functional target.
Area of Science:
- Immunology
- Molecular Biology
- Genetics
Background:
- Immune thrombocytopenia (ITP) is an autoimmune disorder characterized by low platelet counts.
- The role of microRNAs (miRNAs) in ITP pathogenesis is increasingly recognized.
- T helper 17 (Th17) cells are implicated in the immune dysregulation observed in ITP.
Purpose of the Study:
- To investigate the involvement of miRNA-30a in ITP pathogenesis.
- To determine if miRNA-30a affects Th17 cell differentiation in ITP.
- To explore the potential mechanism involving the target gene SOCS3 and its interaction with miRNA-30a.
Main Methods:
- Establishment of a chronic ITP mouse model using anti-mouse platelet serum (APS).
- Detection and correlation analysis of miRNA-30a and RORγt expression in spleen mononuclear cells.
- Luciferase reporter assays, qPCR, and Western blot to verify SOCS3 as a target gene of miRNA-30a.
Main Results:
- The ITP mouse model exhibited significantly decreased platelet counts and elevated miRNA-30a and RORγt expression in spleen mononuclear cells.
- A positive correlation was observed between miRNA-30a and RORγt expression (r=0.54).
- Luciferase assays confirmed binding of miRNA-30a to the SOCS3 3'UTR, but SOCS3 mRNA and protein levels were not significantly altered in ITP mice.
Conclusions:
- The established chronic ITP mouse model successfully demonstrated increased miRNA-30a expression.
- miRNA-30a positively correlates with RORγt, suggesting its role in Th17 cell differentiation and ITP pathogenesis.
- While SOCS3 binds to miRNA-30a, it may not be its functional target gene in the context of ITP.
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