Ultrasound-targeted microbubble destruction-mediated Foxp3 knockdown may suppress the tumor growth of HCC mice by relieving immunosuppressive Tregs function

The aim of the present study was to investigate the effect of Forkhead family transcription factor P3 (Foxp3) knockdown on the function of cluster of differentiation (CD)4(+)CD25(+) regulatory T cell (Tregs) and the tumor growth of a hepatocellular carcinoma (HCC) mouse model. CD4(+)CD25(+) Tregs and CD4(+)CD25(−) T cells were sorted from peripheral blood mononuclear cells (PBMCs) of patients with HCC. Then, ultrasound-targeted microbubble destruction (UTMD)-mediated Foxp3-microRNA (miRNA) was transfected into Tregs. Subsequently, CD4(+)CD25(−) T cells were co-cultured with PBMC and Tregs without Foxp3-miRNA (Foxp3(+)Tregs) or Tregs with Foxp3-miRNA (Foxp3(−)Tregs) and the proliferation-inhibition ratio of CD4(+)CD25(−) T cells was detected using a Cell Counting Kit-8. Additionally, HCC mice were treated with UTMD-mediated Foxp3-shRNA, the tumor volume was calculated and the content of CD4(+) and CD25(+) T cells in the blood were detected using flow cytometry. The content of interferon-γ (IFN-γ), interleukin (IL)-2, IL-10, transforming growth factor-β (TGF-β) and vascular endothelial growth factor (VEGF) in cultural supernatant and serum were detected by ELISA analysis. Foxp3(−)Tregs significantly reduced the inhibition effect of Foxp3(+)Tregs on the proliferation of CD4(+)CD25(−) T cells (P<0.01). The content of IFN-γ and IL-2 significantly increased, while IL-10 and TGF-β significantly decreased in the co-cultured system of Foxp3(−)Tregs compared with the co-cultured system of Foxp3(+)Tregs (P<0.01). Following treatment with Foxp3-shRNA, the average tumor volume, ratio of Tregs/CD4(+) T cells and level of IL-10, TGF-β and VEGF significantly decreased, however, the level of IFN-γ and IL-2 significantly increased compared with un-treated HCC mice (P<0.05). Foxp3 knockdown may suppress the tumor growth of HCC mice through relieving the immunosuppressive function of Tregs.