背景:发现特异性蛋白1(SP1)在调节TGF-β1驱动的上皮-间质转化(EMT)中起关键作用。最近的临床发现表明,随着乳腺癌患者的癌症进展,miR-128-3p的表达显着下降。然而,miR-128-3p对乳腺癌中SP1表达的影响尚不清楚.在这里,我们评估了miR-128-3p模拟物在通过调节TGF-β1/SP1轴抑制乳腺癌细胞系EMT中的作用.
方法:miR-128-3p与SP1的相互作用通过计算机模拟工具和双荧光素酶报告基因测定进行检测。qPCR,westernblot,和免疫细胞化学实验用于确定miR-128-3p和EMT标志物在有和没有miR-128-3p模拟物处理的情况下的表达水平。Further,了解miR-128-3p模拟物对癌症进展的影响,伤口愈合试验等实验,transwell分析,粘附试验,进行细胞周期分析。
结果:在MCF-7和MDA-MB-231细胞系中,SP1和miR-128-3p水平之间存在显著的负相关。miR-128-3p过表达通过抑制SP1核功能阻碍了TGF-β1刺激细胞中SP1介导的EMT标记。Further,用miR-128-3p模拟物处理显着降低了迁移,TGF-β1刺激细胞的侵袭和扩散能力。流式细胞术结果显示miR-128-3p对细胞周期进程有阻碍作用。
结论:首次上调miR-128-3p抑制SP1,从而限制TGF-β1诱导MCF-7和MDA-MB-231细胞系的EMT。这项研究可能为探索新的miRNA疗法根除晚期乳腺癌病例铺平道路。
BACKGROUND: Specificity protein 1 (
SP1) was found to play a critical role in the regulation of TGF-β1 driven epithelial-mesenchymal transition (EMT). Recent clinical findings demonstrated a significant drop in the expression of miR-128-3p with the cancer progression in breast cancer patients. However, the impact of miR-128-3p on the
SP1 expression in breast cancer remains unknown. Herein, we evaluated the role of miR-128-3p mimics in suppressing EMT of breast cancer cell lines by regulating the TGF-β1/SP1 axis.
METHODS: miR-128-3p interaction with
SP1 was detected by in silico tools and dual-luciferase reporter assay. qPCR, western blot, and immunocytochemistry experiments were conducted for determining the expression levels of miR-128-3p and EMT markers with and without the treatment of miR-128-3p mimics. Further, to understand the effect of miR-128-3p mimics on cancer progression, experiments such as wound healing assay, transwell assay, adhesion assay, and cell cycle analysis were performed.
RESULTS: A significant inverse relation between SP1 and miR-128-3p levels was found in MCF-7 and MDA-MB-231 cell lines. miR-128-3p overexpression impeded the SP1 mediated EMT markers in TGF-β1 stimulated cells by inhibiting the
SP1 nuclear function. Further, treatment with miR-128-3p mimics significantly reduced the migration, invasion and spreading capability of TGF-β1 stimulated cells. Flow cytometry results showed the impeding role of miR-128-3p on the cell cycle progression.
CONCLUSIONS: Upregulated miR-128-3p inhibited SP1, thereby limiting the TGF-β1 induced EMT in MCF-7 and MDA-MB-231 cell lines for the first time. This
study may pave the path to explore novel miRNA therapeutics for eradicating advanced breast cancer cases.