Department of Stomatology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, 450052, P. R. China.
Department of Oral and Maxillofacial Surgery, Guanghua School and Hospital of Stomatology; Guangdong Provincial Key Laboratory of Oral Diseases, Sun Yat-sen University, Guangzhou 510055, P. R. China.
Sci Rep. 2017 Apr 20;7:44688. doi: 10.1038/srep44688.
This study aimed to elucidate how microRNA27a-3p (miR-27a-3p) modulates the Wnt/β-catenin signaling pathway to promote the epithelial-mesenchymal transition (EMT) in oral squamous carcinoma stem cells (OSCSCs) by targeting secreted frizzled-related protein 1 (SFRP1). Flow cytometry was used to sort OSCSCs from the SCC-9 and Tca8113 cell lines. The OSCSCs were randomly assigned into the miR-27a-3p inhibitors group, the miR-27a-3p inhibitors-NC group, the si-SFRP1 group, the si-SFRP1 + miR-27a-3p inhibitors group and the blank group. A luciferase reporter, immunofluorescence and Transwell assays were performed to detect luciferase activity, SFRP1, and cell migration and invasion, respectively. The mRNA expression of miR-27a-3p, SFRP1 and EMT markers (E-cadherin, N-cadherin, vimentin and ZEB1) were detected using qRT-PCR. The protein expression of SFRP1, EMT markers and the proteins of the Wnt/β-catenin signaling pathway was detected by Western blotting. OSCSCs showed up-regulated miR-27a-3p, Wnt/β-catenin signaling pathway-related proteins, vimentin, N-cadherin and ZEB1 and down-regulated SFRP1 and E-cadherin. MiR-27a-3p targeted SFRP1. Down-regulated miR-27a-3p resulted in increased E-cadherin and SFRP1 but decreased vimentin, N-cadherin, ZEB1, the Wnt/β-catenin signaling pathway-related proteins, and invasive and migratory cells. Silenced SFRP1 reversed this effect. We found that miR-27a-3p modulated the Wnt/β-catenin signaling pathway to promote EMT in OSCSCs by down-regulating SFRP1.
本研究旨在阐明 microRNA27a-3p(miR-27a-3p)如何通过靶向分泌型卷曲相关蛋白 1(SFRP1)来调节 Wnt/β-catenin 信号通路,从而促进口腔鳞状细胞癌干细胞(OSCSCs)中的上皮-间充质转化(EMT)。流式细胞术用于从 SCC-9 和 Tca8113 细胞系中分选 OSCSCs。将 OSCSCs 随机分为 miR-27a-3p 抑制剂组、miR-27a-3p 抑制剂-NC 组、si-SFRP1 组、si-SFRP1+miR-27a-3p 抑制剂组和空白组。通过荧光素酶报告、免疫荧光和 Transwell 测定分别检测荧光素酶活性、SFRP1 以及细胞迁移和侵袭。使用 qRT-PCR 检测 miR-27a-3p、SFRP1 和 EMT 标志物(E-钙粘蛋白、N-钙粘蛋白、波形蛋白和 ZEB1)的 mRNA 表达。通过 Western blot 检测 SFRP1、EMT 标志物和 Wnt/β-catenin 信号通路蛋白的表达。OSCSCs 表现出上调的 miR-27a-3p、Wnt/β-catenin 信号通路相关蛋白、波形蛋白、N-钙粘蛋白和 ZEB1,以及下调的 SFRP1 和 E-钙粘蛋白。miR-27a-3p 靶向 SFRP1。下调 miR-27a-3p 导致 E-钙粘蛋白和 SFRP1 增加,但波形蛋白、N-钙粘蛋白、ZEB1、Wnt/β-catenin 信号通路相关蛋白以及侵袭和迁移细胞减少。沉默 SFRP1 逆转了这一效应。我们发现,miR-27a-3p 通过下调 SFRP1 来调节 Wnt/β-catenin 信号通路,从而促进 OSCSCs 中的 EMT。