Wu Fang, Li Jun, Guo Ni, Wang Xiao-Hui, Liao Yu-Qian
Department of Oncology, The First Affiliated Hospital of Nanchang University Nanchang 330006, P. R. China.
Department of Radiation Oncology, Jiangxi Cancer Hospital Nanchang 330029, P. R. China.
Am J Cancer Res. 2017 Mar 1;7(3):405-416. eCollection 2017.
This study aims to elucidate the effects of microRNA-27a (miR-27a) on the proliferation and invasion of gastric cancer (GC) cells by targeting SFRP1 via Wnt/β-catenin signaling pathway. GC and normal adjacent tissues were collected from 273 GC patients. Human gastric cancer cell line (MGC803) and normal human gastric mucosal cell line (GES-1) were cultured. The miR-27a mRNA expression was analyzed using Quantitative real-time polymerase chain reaction (qRT-PCR). Immunohistochemistry (IHC) test was used to detect miR-27a and SFRP1 protein expressions. After transfection, cells were divided into five groups: the negative control (NC) group, the miR-27a inhibitor group, the miR-27a mimics group, the miR-27a inhibitor + siRNA group and the miR-27a mimics + SFRP1 overexpression group. Western blotting was conducted to test SFRP1 and Wnt/β-catenin protein expression. Analysis for the target gene of miR-27a was performed using Luciferase assay. Cell proliferation, migration and invasion were determined by CCK8 and Transwell assay. The dual-luciferase reporter assay system was applied to analyze the effects of miR-27a on Wnt/β-catenin signaling pathway. In GC tissue and cell line, miR-27a protein and mRNA expressions were up-regulated, and SFRP1 protein and mRNA expressions were down-regulated. Luciferase assay indicated that miR-27a might target and regulate its expressions. When miR-27a was down-regulated, SFRP1 was up-regulated, and β-catenin, Wnt, p-β-catenin, and p-Wnt were significantly down-regulated. Compared with the NC group, the proliferation, migration and invasion of GC cells were remarkably increased in the miR-27a group, but these were declined in the miR-27a mimics + SFRP1 overexpression group. The proliferation, migration and invasion of GC cells were elevated in the miR-27a inhibitor + SFRP1 siRNA group compared with the miR-27a inhibitor group. These results showed that miR-27a was highly expressed in GC tissues and cells, and it might promote cell proliferation, migration and invasion by targeting via the activation of Wnt/β-catenin signaling pathway.
本研究旨在通过Wnt/β-连环蛋白信号通路靶向SFRP1，阐明微小RNA-27a（miR-27a）对胃癌（GC）细胞增殖和侵袭的影响。收集了273例GC患者的GC组织及癌旁正常组织。培养人胃癌细胞系（MGC803）和人正常胃黏膜细胞系（GES-1）。采用定量实时聚合酶链反应（qRT-PCR）分析miR-27a mRNA表达。免疫组织化学（IHC）检测用于检测miR-27a和SFRP1蛋白表达。转染后，细胞分为五组：阴性对照（NC）组、miR-27a抑制剂组、miR-27a模拟物组、miR-27a抑制剂 + siRNA组和miR-27a模拟物 + SFRP1过表达组。进行蛋白质免疫印迹法检测SFRP1和Wnt/β-连环蛋白蛋白表达。使用荧光素酶报告基因检测法对miR-27a的靶基因进行分析。通过CCK8和Transwell检测法测定细胞增殖、迁移和侵袭能力。应用双荧光素酶报告基因检测系统分析miR-27a对Wnt/β-连环蛋白信号通路的影响。在GC组织和细胞系中，miR-27a蛋白和mRNA表达上调，SFRP1蛋白和mRNA表达下调。荧光素酶报告基因检测表明miR-可能靶向并调节其表达。当miR-27a下调时，SFRP1上调，β-连环蛋白、Wnt、p-β-连环蛋白和p-Wnt显著下调。与NC组相比，miR-27a组GC细胞的增殖、迁移和侵袭明显增加，但在miR-27a模拟物 + SFRP1过表达组中这些能力下降。与miR-27a抑制剂组相比，miR-27a抑制剂 + SFRP1 siRNA组GC细胞的增殖、迁移和侵袭能力增强。这些结果表明，miR-27a在GC组织和细胞中高表达，它可能通过激活Wnt/β-连环蛋白信号通路靶向促进细胞增殖、迁移和侵袭。
