METHODS: Different formononetin concentrations (0, 20, 40, 60, 80, 100 μM) were used to treat BC cells transfected with pcDNA3.1-Programmed death ligand 1 (PD-L1) or Short-hairpin RNA (sh)-PD-L1. Cells were separated into four subgroups: CTRL, pcDNA3.1-PD-L1, sh-CTRL, and sh-PD-L1. Cell viability and cell cycle were assessed through Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay and flow cytometry. Programmed death ligand 1 (PD-L1) mRNA concentration was validated via quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). Cell metastasis was evaluated via cloning assay and transwell assay. The p-STING/stimulator of interferon genes (STING), p-p65/p65, and PD-L1 concentrations were determined by western blot.
RESULTS: Formononetin restrained the proliferation of MCF-7 and MDA-MB-468 cells, and reduced PD-L1 mRNA, p-STING/STING, and p-p65/p65 protein concentrations. Whereas PD-L1 inhibition restrained the viability of BC cells, pcDNA3.1-PD-L1 intervention had the opposite result. STING pathway inhibitor C-176 combined with formononetin treatment further restrained cell proliferation, colony formation, and cell invasion, in contrast to cells treated with formononetin alone.
CONCLUSIONS: Formononetin can restrain the proliferation of BC cells, which may be mediated through the interference of PD-L1 and suppression of the activation of the STING-NF-κB pathway.
方法:使用不同浓度(0、20、40、60、80、100μM)处理用pcDNA3.1-程序性死亡配体1(PD-L1)或短发夹RNA(sh)-PD-L1转染的BC细胞。细胞分为四个亚组:CTRL,pcDNA3.1-PD-L1,sh-CTRL,和sh-PD-L1.通过甲基噻唑基二苯基-溴化四唑(MTT)测定和流式细胞术评估细胞活力和细胞周期。通过定量实时逆转录聚合酶链反应(qRT-PCR)验证程序性死亡配体1(PD-L1)mRNA浓度。通过克隆测定和transwell测定评估细胞转移。干扰素基因的p-STING/刺激因子(STING),通过蛋白质印迹测定p-p65/p65和PD-L1浓度。
结果:Formononetin抑制MCF-7和MDA-MB-468细胞的增殖,减少PD-L1mRNA,p-STING/STING,和p-p65/p65蛋白浓度。而PD-L1抑制抑制了BC细胞的活力,pcDNA3.1-PD-L1干预具有相反的结果。STING通路抑制因子C-176联合海蒙素处置进一步克制细胞增殖,菌落形成,和细胞入侵,与单独用海蒙素处理的细胞相反。
结论:Formononetin能抑制BC细胞的增殖,这可能是通过干扰PD-L1和抑制STING-NF-κB通路的激活来介导的。