Promotion of miR-143-3p on Treg cells differentiation in vitro
As previously mentioned, the expression of miR-143-3p has been associated with Treg cells differentiation in RA, which considered as a critical regulator of peripheral immune tolerance and homeostasis[6]. Hence, to directly investigate the potential effect of miR-143-3p on the differentiation of Treg cells, we cultured the naïve CD4+T cells from C57BL/6 mice under induced Treg (iTreg) cell–polarizing conditions in vitro. As controls, naive CD4+T cells were activated with anti-CD3ε/CD28 antibodies without the addition of differentiating cytokines (Th0 cells). We found that miR-143-3p mRNA expression was up-regulated in Treg cells differentiation when compared with Th0 (Figure 6A). Meanwhile, miR-143-3p shRNAs and mock shRNAs were infected into naïve CD4+ T cells, and then cultured in the condition of Th17-polarization. The successful infection, which significantly altered the expression of the intracellular miR-143-3p, was verified by qPCR (Figure 6B). The overexpression of miR-143-3p markedly elevated the mRNA levels of forkhead box P3 (Foxp3), which was the master transcription factor of Treg cells. In contrast, expression of Foxp3 mRNA was notably reduced in the down-regulated cell population (Figure 6C). This funding indicates that miR-143-3p is capable to facilitate Treg cells differentiation in vitro.