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Background  Previous studies reported that various miRNAs participate in autoimmune diseases, but the potential regulatory mechanism of miRNAs in autoimmune thyroiditis (AIT) needs further exploration.    Objective  This study aimed to further verify that miR-326 contributes to AIT by regulating Th17/Treg balance through Ets-1 using lentiviral gene delivery through tail vein and thyroid injection in NOD.H-2 h4  mice.    Materials and Methods  Five-week-old NOD.H-2 h4  mice were divided randomly into tail vein and thyroid injection groups, and each received either mmu-miR-326 sponge (LV-sponge) or lentiviral vector control. Mice were divided for tail vein injection: the therapeutic LV-ctrl, therapeutic LV-sponge, prophylactic LV-ctrl, and prophylactic LV-sponge groups. The control group was fed high-iodine water without vein injection. The thyroid infiltration of lymphocytes and serum TgAb value were investigated by thyroid hematoxylin and eosin (HE) staining and ELISA, respectively. Ets-1 and lymphocyte counts were measured by RT-PCR, western blotting, and flow cytometry. The thyroid CD4 + IL-17a +  cells and CD4 + Ets-1 +  cells were detected by immunofluorescence, and the serum cytokines were tested by ELISA.    Results  In the tail vein injection groups, the thyroid inflammatory score and serum TgAb titer were significantly lower in the LV-sponge groups than in the control and LV-ctrl groups while Ets-1 protein expression in mouse spleens was increased in the LV-sponge groups. Moreover, Th17/Treg ratio declined in the LV-sponge group and decreased significantly in the prophylactic LV-sponge group ( P  = 0.036) tested by flow cytometry. Immunofluorescence showed that, in LV-sponge groups, CD4 + IL-17a +  cells were decreased significantly ( P  = 0.001), while CD4 + Ets-1 +  cells were increased significantly in the LV-sponge group ( P  = 0.029). The serum IL-17/IL-10 was decreased significantly in the LV-sponge group ( P  < 0.05). In the thyroid injection groups, the thyroid inflammatory score and serum TgAb titer in the LV-sponge group decreased significantly compared with those in the LV-ctrl group ( P  < 0.05). In addition, in LV-sponge groups, CD4 + IL-17a +  cells were decreased, while CD4 + Ets-1 +  cells were increased significantly in the inhibition group evaluated by immunofluorescence. Moreover, tail vein injection of LV-sponge resulted in much lower TgAb levels in thyroiditis compared with thyroid injection.    Conclusion  MiR-326 targeted therapy may be a promising approach for AIT. In addition, tail vein injection may achieve a better intervention effect than thyroid injection.

In Vivo Inhibition of MicroRNA-326 in a NOD.H-2h4 Mouse Model of Autoimmune Thyroiditis