Department of Medical Oncology, Yantaishan Hospital, Yantai, Shandong 264000, P.R. China.
Department of Radiotherapy, Yidu Central Hospital of Wei Fang, Qingzhou, Shandong 262500, P.R. China.
Int J Mol Med. 2020 Nov;46(5):1755-1764. doi: 10.3892/ijmm.2020.4720. Epub 2020 Sep 4.
It has been reported that microRNA (miRNA/miR)‑181b plays an important role in regulating cellular proliferation, invasion and apoptosis in various tumors. However, the role of miR‑181b and its molecular mechanisms in colon cancer cells have not yet been elucidated. The present study thus aimed to investigate the mechanisms of miR‑181b targeting cylindromatosis (CYLD) to regulate the nuclear factor‑κB (NF‑κB) signaling pathway, and to determine its role in colon cancer cell proliferation and apoptosis. For this purpose, miR‑181b was overexpressed and silenced in the SW480 cell line. The cell proliferation and apoptotic rates were determined using a Cell Counting kit and colony formation assays, and Annexin V‑FITC staining, respectively. The expression levels of proteins associated with the NF‑κB signaling pathway and apoptosis were detected by western blot analysis. Furthermore, a dual luciferase assay was applied to confirm the interaction between miR‑181b and CYLD. CYLD was also overexpressed and silenced in the SW480 cell line using a CYLD overexpression plasmid and siRNA technology, respectively. Transfected cells were used for subsequent experiments. In addition, a nude mouse model was established to measure tumor volume and weight. Immunohistochemistry and a TUNEL assay were performed to detect the Ki67 levels and the cell apoptotic rate, respectively. Compared with the control group, miR‑181 silencing or CYLD overexpression significantly attenuated cell proliferation, invasion and migration, and notably increased the proportion of apoptotic cells. Furthermore, the expression levels of Bax and cleaved caspase‑3 were markedly increased, whereas those of Bcl‑2 were significantly decresaed (P<0.05). In addition, the protein expression levels of p‑p65/p65 and p‑IκBα/IκBα were significantly downregulated and upregulated, respectively (P<0.05). Consistent with the results obtained in vitro, in vivo experiments using a nude mouse model yielded similar findings. The aforementioned results indicated that miR‑181b downregulation inhibited human colon cancer cell proliferation by targeting CYLD to attenuate the activity of the NF‑κB signaling pathway.
据报道，微小 RNA（miRNA/miR）-181b 在各种肿瘤中细胞增殖、侵袭和凋亡中发挥重要作用。然而，miR-181b 在结肠癌细胞中的作用及其分子机制尚未阐明。因此，本研究旨在探讨 miR-181b 靶向 CYLD 以调节核因子-κB（NF-κB）信号通路的机制，并确定其在结肠癌细胞增殖和凋亡中的作用。为此，在 SW480 细胞系中过表达和沉默 miR-181b。使用细胞计数试剂盒和集落形成实验分别测定细胞增殖率和凋亡率，并用 Annexin V-FITC 染色进行检测。通过 Western blot 分析检测与 NF-κB 信号通路和凋亡相关的蛋白表达水平。此外，应用双荧光素酶报告基因实验来验证 miR-181b 与 CYLD 之间的相互作用。还通过 CYLD 过表达质粒和 siRNA 技术分别在 SW480 细胞系中过表达和沉默 CYLD。转染细胞用于后续实验。此外，建立裸鼠模型以测量肿瘤体积和重量。通过免疫组化和 TUNEL 检测分别检测 Ki67 水平和细胞凋亡率。与对照组相比，miR-181b 沉默或 CYLD 过表达显著抑制细胞增殖、侵袭和迁移，显著增加凋亡细胞的比例。此外，Bax 和 cleaved caspase-3 的表达水平明显增加，而 Bcl-2 的表达水平明显降低（P<0.05）。此外，p-p65/p65 和 p-IκBα/IκBα 的蛋白表达水平显著下调和上调（P<0.05）。与体外结果一致，裸鼠模型的体内实验也得到了类似的结果。上述结果表明，miR-181b 通过靶向 CYLD 抑制人结肠癌细胞增殖，从而抑制 NF-κB 信号通路的活性。
