Background Systemic lupus erythematosus (SLE) patients showed an imbalance between CD4+CD25+FoxP3+ T regulatory (Treg) and IL-17-producing cells (Th17), as well as the deficient activity of bone marrow mesenchymal stem cells (MSCs). The expression imbalance of microRNAs(miRNAs) can cause cell dysfunction, which participates in the pathogenesis of SLE. However, how miRNAs regulate MSCs activity in lupus is not clear.
Objectives The aim of this study is to investigate whether miR-663 participates in SLE MSCs immune modulation by directly down-regulating TGF-β1 mRNA, which might contribute to Treg/Th17 imbalancein lupus, to further understand the pathogenesis of SLE.
Methods BMSCs were isolated, cultured and expanded from iliac crest bone marrow of healthy donors and SLE patients. MicroRNA expressions of BMSCs were determined by MicroRNA array analysis. Real-time PCR was used to further determine the miR-663 expressionand TGF-β1 mRNAlevel. The important computer predicted target of mir-663, TGF-β1 was determined using the luciferase reporter assay system. Pre-miR-663a, miRNA control, and anti-miR-663a were obtained from Invitrogen. Transfection of MSCs with miRNAs was done by using Lipofectamine2000. BMSCs from healthy controls and SLE patients were co-cultured with PBMCs from SLE patients for 24 hours, and flow cytometry were used to detect their effect on the ratio of Treg/Th17.
Results Mir-663 and mir-638 expressions were significantly up-regulated(2.52- fold and 2.34-fold higher individually, n=4, P<0.05) and let-7f expression was markedly down-regulated(2.03- fold lower, n=4, P<0.05) in MSCs from SLE patients compared to normal controls. The expression of miR-663 was markedly up-regulated in lupus MSCs by real-time PCR (1.43- fold higher, n=4, P<0.05), and the synthesis of TGF-β1 mRNAs was significantly lower(3.88- fold lower, n=4, P<0.05). A fluorescence microscope showed that the miRNAs transfection efficiency is about 52%>64%. Transfection of SLE MSCs with pre-miR-663a caused significant upregulation of miR-663a expression(8.33- fold higher, n=3, P<0.01) and markedly lower synthesis of TGF-β1 mRNA(1.52-fold lower, n=3, P<0.05), while transfection with anti-miR-663a caused significant downregulation of miR-663a expression(3.01- fold lower, n=3, P<0.05), as compared to normal controls. MiR-663a transfected MSCs maintained similar cell morphology and phenotypic surface antigens of MSCs. The mean value of Treg/Th17was significantly decreased in PBMCs from SLE patients compared to normal controls(0.48±0.12vs0.65±0.09, n=6, P<0.01). Compared to SLE MSCs, MSCs from normal controls exhibited a better immune suppression effect through up-regulating Treg/Th17of SLE patients(0.68±0.15vs0.54±0.14, n=3, P<0.05), and transfection of normal MSCs with pre-miR-663a caused significant downregulation of Treg/Th17 (0.38±0.07vs0.68±0.15, n=3, P<0.01), while transfection with anti-miR-663a led to an opposite effect(0.76±0.08vs0.68±0.15, n=3, P<0.05).
Conclusions SLE patients show an imbalance betweenTregand Th17 cells, whichmight be associated with down-regulation of TGF-β1 in bone marrow-derived mesenchymal stem cells that mediated by miR-663.
Disclosure of Interest None Declared