Department of Oncology, The First Affiliated Hospital of Chongqing Medical University, Yuzhong, Chongqing 400016, P.R. China.
Oncol Rep. 2017 Nov;38(5):3144-3152. doi: 10.3892/or.2017.5968. Epub 2017 Sep 19.
Interleukin-8 (IL-8), which is secreted by cancer cells undergoing epithelial-mesenchymal transition (EMT), can promote EMT in adjacent epithelial-like cells. MicroRNAs (miRNAs/miRs) can affect the expression of target genes via binding to their 3'-untranslated regions (3'-UTRs), which may subsequently affect the biological behaviors of cancer cells. In our previous study, miR-520c-3p was predicted to directly target the 3'-UTR of IL-8. Therefore, the present study was carried out to investigate whether miR-520c-3p can interact with the IL-8 gene and regulate the EMT of breast cancer cells. Web-based prediction algorithms were used to identify miRNAs that potentially target the IL-8 transcript. Luciferase reporter assays were used to confirm the targeting of IL-8 by miR-520c-3p. Reverse transcription-quantitative PCR and western blot analyses were used to examine the levels of IL-8 and EMT-related genes in breast cancer cells. The functional impact of miR-520c-3p on EMT phenotype was evaluated using Transwell and wound-healing assays, and rescue experiments were conducted by overexpressing IL-8 to determine its effect on cell properties. miR-520c-3p was predicted by all three databases, which strongly suggested its interaction with the 3'-UTR of IL-8. The relative Renilla luciferase activity of luciferase reporter construct containing the wild-type 3'-UTR of IL-8 was markedly decreased by miR-520c-3p transfection when compared with scrambled miRNA control transfection (P<0.001). In addition, compared with the scrambled miRNA control transfection, the overexpression of miR-520c-3p significantly reduced the expression of IL-8, and resulted in increased E-cadherin and decreased vimentin and fibronectin levels in MCF-7 and T47D cells (all P<0.001). Introduction of miR-520c-3p inhibited the invasion and migration of MCF-7 and T47D cells (all P<0.001). By contrast, the rescue of IL-8 expression led to the recovery of EMT-related protein expression patterns and cell motility and invasion capabilities. In conclusion, aberrant miR-520c-3p expression may lead to reduced IL-8 expression and promote the mesenchymal phenotype in breast cancer cells, thereby increasing invasive growth.
白细胞介素 8（IL-8）是一种由发生上皮间质转化（EMT）的癌细胞分泌的细胞因子，它可以促进邻近上皮样细胞中的 EMT。微小 RNA（miRNA/miRs）可以通过与靶基因的 3'非翻译区（3'-UTR）结合来影响靶基因的表达，从而可能随后影响癌细胞的生物学行为。在我们之前的研究中，miR-520c-3p 被预测可以直接靶向 IL-8 的 3'-UTR。因此，本研究旨在探讨 miR-520c-3p 是否可以与 IL-8 基因相互作用并调节乳腺癌细胞的 EMT。使用基于网络的预测算法来识别可能靶向 IL-8 转录本的 miRNA。使用荧光素酶报告基因检测来验证 miR-520c-3p 对 IL-8 的靶向作用。使用逆转录-定量 PCR 和 Western blot 分析来检测乳腺癌细胞中 IL-8 和 EMT 相关基因的水平。使用 Transwell 和划痕愈合试验评估 miR-520c-3p 对 EMT 表型的功能影响，并通过过表达 IL-8 进行挽救实验以确定其对细胞特性的影响。所有三个数据库均预测 miR-520c-3p 与 IL-8 的 3'-UTR 相互作用。与对照 scrambled miRNA 转染相比，含有野生型 IL-8 3'-UTR 的荧光素酶报告基因构建体的相对 Renilla 荧光素酶活性明显降低（P<0.001）。此外，与对照 scrambled miRNA 转染相比，miR-520c-3p 的过表达显著降低了 MCF-7 和 T47D 细胞中 IL-8 的表达，导致 E-钙粘蛋白水平升高，波形蛋白和纤连蛋白水平降低（均 P<0.001）。miR-520c-3p 的转染抑制了 MCF-7 和 T47D 细胞的侵袭和迁移（均 P<0.001）。相反，IL-8 表达的挽救导致 EMT 相关蛋白表达模式和细胞迁移和侵袭能力的恢复。总之，异常表达的 miR-520c-3p 可能导致 IL-8 表达减少并促进乳腺癌细胞中的间充质表型，从而增加侵袭性生长。
