Inhibition of GATA3 Expression Upon miR-29b1 Overexpression in CD4+ Cells

Background and Aim: MicroRNAs (miRNAs) are about 18-24 endogenous nucleotides non-coding RNAs. These short RNAs regulategene expression by binding to 3 -untranslated region of mRNA that ledto mRNA degradation or translation inhibition. Transcription factors areone of the most important genes that their expression is controlled bymiRNAs. Several studies showed that GATA3 is the master regulator ofTh2 differentiation. Th2 have a key role in allergy and hypersensitivity.The aim of this study was to determine whether miR-29b overexpressioncould modulate the expression of the GATA3 transcription factor inCD4+ cells.Methods: Spleens were harvested from 6-8 weeks old C57Bl/6 femalemice and single cells suspension was prepared. CD4+ cells were thenpurified by negative selection on MACS separator (Miltenyi Biotec).The purity of cells was confirmed by flow cytometry. Then, CD4+ cellswere cultured at a density of 106 cells/well in 24-well culture platesand stimulated with anti-CD3/CD28 mAbs. After 4 hours, the cellswere transfected with miR-29b1 mimic using Lipofectamine 2000. 48hours after transfection, total RNA was extracted and Quantitative realtimePCR was performed. Expression of mature miR-29b1 and GATA3were normalized to U47 and β-actin, respectively, as the endogenousreference gene, and relative expression was calculated using the 2-ΔΔctmethod.Results: To investigate the effect of miR-29b1 overexpression on GATA3expression, miR-29b1 mimic was transfected into the purified mouseCD4+ cells. RNA extraction and Quantitative real-time PCR revealed thatmiR-29b1 mimic was successfully transferred to CD4+ cells. Furthermore,GATA3 expression was significantly decreased in miR-29b1 transfectedcells compared to control cells.Conclusion: Here, we showed that overexpression of miR-29b1 decreasedGATA3 expression in CD4+ cells. Since this transcription factor has akey function in the differentiation of T naive to T cell subsets, miR-29b1overexpression could inhibit Th2 differentiation in allergic diseases.

Keywords:

miRNA , Lymphocytes , Th2 , GATA3 , Differentiation