慢病毒介导RNA抑制黑素瘤细胞Foxp3蛋白表达

目的:利用RNA干扰(RNAi)技术在体外干扰小鼠B16黑素瘤细胞Foxp3基因表达,探讨RNA干扰用于黑素瘤治疗的可行性。方法:针对Foxp3基因设计小干扰RNA(siR-NA),构建起短发夹状RNA(shRNA)慢病毒表达载体,并转染小鼠B16细胞,在体外诱导RNA干扰。分别采用Westernblot和RT-PCR检测Foxp3基因的表达情况;ELISA检测TGF-β1、TGF-β2和IL-10等细胞因子的变化;将干扰后的小鼠B16细胞与CD4+CD25-T淋巴细胞共培养,CCK8法检测CD4+CD25-T淋巴细胞增殖能力。结果:通过Foxp3 shRNA的转染,可实现对B16细胞Foxp3表达的沉默,可下调肿瘤细胞对CD4+CD25-T淋巴细胞增殖抑制的能力,并且下调TGF-β1、TGF-β2和IL-10等细胞因子的表达,尤其是TGF-β2的表达。结论:RNA干扰可抑制小鼠黑素瘤细胞靶基因Foxp3的表达及细胞增殖,并对CD4+CD25-T淋巴细胞增殖抑制的能力减弱,同时减弱抑制性细胞因子的分泌,为黑素瘤的基因治疗提供了新思路。

Inhibitory effect of lentiviral-mediated RNA on the expression of Foxp3 protein in melanoma cells

AIM: To explore the feasibility of RNA interference in the treatment of melanoma by inhibiting the Foxp3 gene expression in mouse B16 melanoma cells using RNA interference(RNAi) in vitro.METHODS: Small interfering RNA(siRNA) was designed according to Foxp3 gene.A short hairpin RNA(shRNA) lentivirus expression vector was constructed and transfected into mouse B16 cells,and RNA interference was induced in vitro.Western blot and real-time RT-PCR were performed to detect the expression of Foxp3 gene.ELISA was applied to detect the changes of TGF-β1,TGF-β2,IL-10 and other cytokines.The B16 cells after interference were co-cultured with CD4+CD25-T lymphocytes.CCK8 assay was used to monitor the proliferation of CD4+CD25-T lymphocytes.RESULTS: shRNA could suppress the expression level of Foxp3,down-regulate the inhibitory ability of tumor cells on the proliferation of CD4+CD25-T lymphocytes,and reduce the secretion of TGF-β1,TGF-β2,IL-10 and other cytokines,in particular the expression of TGF-β2.CONCLUSION: RNA interference can inhibit the expression of target gene Foxp3 in mice melanoma cells and the proliferation of tumor cells.It can also reduce the inhibition on the proliferation of CD4+CD25-T lymphocytes,and the secretion of inhibitory cytokines.