Department of Surgical Emergency, The First Affiliated Hospital of Fujian Medical University, Fuzhou, Fujian 350005, P.R. China.
Mol Med Rep. 2019 Oct;20(4):3735-3745. doi: 10.3892/mmr.2019.10597. Epub 2019 Aug 21.
The aberrant expression of tumor suppressor Smad4 often occurs in colorectal cancer (CRC), and this phenomenon is believed to be associated with drug resistance. The present study aimed to investigate the effects of Smad4 on the sensitivity of CRC cells to cetuximab, and the possible mechanism underlying such an effect. A total of 629 colorectal adenocarcinoma cases were downloaded from The Cancer Genome Atlas (TCGA) database, and a Smad4 mutation rate of ~21% was demonstrated among the cases. Low expression of Smad4 was present in CRC tissues analyzed by TCGA and in four CRC cell lines, as determined by reverse transcription‑quantitative PCR (RT‑qPCR) and western blot analysis. Cell Counting kit‑8 (CCK‑8) was used to measure the effects of different concentrations of cetuximab on SW480 cell viability at 24 and 48 h. The results demonstrated that treatment of SW480 cells with 20 µg/ml cetuximab for 48 h markedly reduced cell viability. In addition, plasmids were transfected into SW480 cells to induce Smad4 silencing or overexpression. Silencing Smad4 attenuated the sensitivity of SW480 CRC cells to cetuximab; this effect was reflected in increased cell viability and slightly increased migration and invasion, as determined by CCK‑8, wound scratch and Transwell analyses. RT‑qPCR and western blotting was performed to assess the expression levels of apoptosis‑ and epithelial‑mesenchymal transition (EMT)‑related genes. Silencing Smad4 partly reversed the effects of cetuximab on the mRNA and protein expression levels of vimentin, Bax/Bcl‑2 and E‑cadherin. However, Smad4 overexpression enhanced SW480 cell sensitivity to cetuximab. In conclusion, Smad4 may serve a vital role in the sensitivity of CRC cells to chemotherapeutic drugs by promoting EMT.
抑癌基因 Smad4 的异常表达常发生于结直肠癌（CRC）中，且这种现象被认为与耐药性有关。本研究旨在探讨 Smad4 对 CRC 细胞对西妥昔单抗敏感性的影响及其可能的作用机制。从癌症基因组图谱（TCGA）数据库中下载了 629 例结直肠腺癌病例，其中约 21%的病例存在 Smad4 突变。通过 TCGA 和四个 CRC 细胞系的逆转录-定量 PCR（RT-qPCR）和 Western blot 分析，发现 Smad4 在 CRC 组织中低表达。细胞计数试剂盒-8（CCK-8）用于测量不同浓度西妥昔单抗对 SW480 细胞活力在 24 和 48 h 的影响。结果表明，用 20 μg/ml 西妥昔单抗处理 SW480 细胞 48 h 可明显降低细胞活力。此外，将质粒转染到 SW480 细胞中以诱导 Smad4 沉默或过表达。沉默 Smad4 可减弱 SW480 CRC 细胞对西妥昔单抗的敏感性；通过 CCK-8、划痕和 Transwell 分析测定，细胞活力增加，迁移和侵袭略有增加，可反映这一作用。RT-qPCR 和 Western blot 用于评估凋亡和上皮-间充质转化（EMT）相关基因的表达水平。沉默 Smad4 部分逆转了西妥昔单抗对波形蛋白、Bax/Bcl-2 和 E-钙粘蛋白的 mRNA 和蛋白表达水平的影响。然而，Smad4 过表达增强了 SW480 细胞对西妥昔单抗的敏感性。综上所述，Smad4 可能通过促进 EMT 在 CRC 细胞对化疗药物的敏感性中发挥重要作用。
