Department of Surgical Oncology, North China University of Science and Technology Affiliated Hospital, Tangshan, China.
Department of General Surgery, North China University of Science and Technology Affiliated Hospital, Tangshan, China.
J Physiol Pharmacol. 2022 Apr;73(2). doi: 10.26402/jpp.2022.2.07. Epub 2022 Sep 29.
This study aimed to investigate the effect of neuropilin-1 (NRP-1) silencing on epithelial-mesenchymal transformation (EMT) mediated by transforming growth factor-β1 (TGF-β1) and on the proliferation and migration of colon cancer SW480 cells. After transfection of small interfering ribonucleic acid (siRNA)-NRP-1 into colon cancer SW480 cells, the messenger RNA (mRNA) and protein expression levels of NRP-1 were detected using quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. Four EMT models were induced using 0, 2, 5, and 10 ng/mL TGF-β1, respectively. Cell proliferation was detected using Cell Counting Kit-8, and the protein levels of EMT markers E-cadherin and vimentin were detected using Western blot. EMT was induced in the transfected SW480 cells using TGF-β1, after which four groups were created: a negative control group (siRNA-Ncontrol), a transfection group (siRNA-NRP-1), an induction group (TGF-β1), and a transfection + induction group (siRNA-NRP-1+TGF-β1). Western blot was then used to detect the expression of E-cadherin and vimentin, and cell proliferation and migration were detected using cell counting kit-8 (CCK-8) and scratch assay. After transfection with siRNA-NRP-1, the mRNA and protein expression levels of SW480 cells were significantly decreased (P<0.05). After 48 hours of induction with 10 ng/mL TGF-β1, cell proliferation was obvious, E-cadherin expression decreased, and vimentin expression significantly increased (P<0.05), indicating that EMT had been successfully induced compared with the induction group, the transfection + induction group had significantly increased E-cadherin expression after corresponding treatments (including transfection and induction alone) (P<0.05), and the proliferation and migration of colon cancer cells decreased (P<0.05). In conclusion: silencing, NRP-1 in colon cancer SW480 cells can partially reverse TGF-β1-mediated EMT, reduce the proliferation activity of colon cancer cells, and slow their migration ability. Therefore, NRP-1 may become a new target for the treatment of colon cancer.
本研究旨在探讨神经纤毛蛋白-1(NRP-1)沉默对转化生长因子-β1(TGF-β1)介导的上皮-间充质转化(EMT)以及结肠癌细胞 SW480 增殖和迁移的影响。用小干扰核糖核酸(siRNA)-NRP-1 转染结肠癌细胞 SW480 后,采用实时定量聚合酶链反应(qRT-PCR)和 Western blot 检测 NRP-1 的信使 RNA(mRNA)和蛋白表达水平。分别用 0、2、5 和 10 ng/mL TGF-β1 诱导 4 种 EMT 模型。采用细胞计数试剂盒-8(Cell Counting Kit-8,CCK-8)检测细胞增殖,Western blot 检测 EMT 标志物 E-钙黏蛋白和波形蛋白的蛋白水平。用 TGF-β1 诱导转染的 SW480 细胞发生 EMT,然后创建 4 个组:阴性对照组(siRNA-Ncontrol)、转染组(siRNA-NRP-1)、诱导组(TGF-β1)和转染+诱导组(siRNA-NRP-1+TGF-β1)。然后用 Western blot 检测 E-钙黏蛋白和波形蛋白的表达,用 CCK-8 和划痕实验检测细胞增殖和迁移。转染 siRNA-NRP-1 后,SW480 细胞的 mRNA 和蛋白表达水平显著降低(P<0.05)。用 10 ng/mL TGF-β1 诱导 48 小时后,细胞增殖明显,E-钙黏蛋白表达减少,波形蛋白表达显著增加(P<0.05),表明与诱导组相比 EMT 成功诱导,转染+诱导组在单独转染和诱导后 E-钙黏蛋白表达显著增加(P<0.05),结肠癌细胞的增殖和迁移减少(P<0.05)。结论:沉默结肠癌细胞 SW480 中的 NRP-1 可部分逆转 TGF-β1 介导的 EMT,降低结肠癌细胞的增殖活性,减缓其迁移能力。因此,NRP-1 可能成为结肠癌治疗的新靶点。