Overexpression of HIPK3 memorably attenuated miR-146a-mediated downregulation of p-STAT3 and suppression of inflammation in TGF-β-induced thymocytes. TGF-β-induced thymocytes were transfected with miR-146a mimics, HIPK3 siRNAs or MiR-146a mimics + HIPK3-overexpressed plasmids. (A) Changes of miR-146a and HIPK3 expressions in each group were evaluated via RT-qPCR assay. (B) Western blot assay was carried out to analyze the expression of HIPK3, FOXP3 and p-STAT3 in each group. (C) The relative expression of FOXP3 in each group was determined by RT-qPCR assay. (D) IF assay exhibited the co-localization and expression of HIPK3 and STAT3 in the processed thymocytes. Magnification, 400×, scale bar =20 µm. (E) After transfection, the levels of IL-10, TGF-β, IL-4, and TNF-α in thymocytes induced by TGF-β were detected by ELISA assay. *P<0.05, **P<0.01 vs. NC group; #P<0.05 vs. mimics group. NC, negative control; HIPK3, homeodomain-interacting protein kinases 3; TGF-β, transforming growth factor-β; siRNA, small interfering RNA; RT-qPCR, reverse transcription quantitative polymerase chain reaction; IF, immunofluorescence; IL-10, interleukin 10; IL-4, interleukin 4; TNF-α, tumor necrosis factor-α; ELISA, enzyme-linked immunosorbent assay.