Department of Gastrointestinal Surgery, First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu, People's Republic of China.
J Cancer Res Clin Oncol. 2012 May;138(5):763-74. doi: 10.1007/s00432-012-1154-x. Epub 2012 Jan 22.
The aim of this study was (a) to determine the role of micro-223 (miR-223) in gastric cancer and (b) to elucidate its regulatory mechanism on the FBXW7/hCdc4 gene.
Artificial miR-223 and control oligonucleotide was transfected into gastric cancer cell line SGC7901 by using Lipofectamine2000. Apoptosis of miR-223 group and control group cells was analyzed by flow cytometry, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide and colony formation assays were performed to detect the cell viability, to survey migration of miR-223 group and control group cells; scratch wound-healing motility assays, Transwell Assay, and Western blot test were performed to measure the variance of hFBXW7. Luciferase Reporter Assay, which was done by pLUC-hFBXW7 WT-3'-UTR co-transfected with pLMP-hsa-miR-223 or pLMP plasmid (as control) into HEK293T cells, used to detect whether hFBXW7 is a direct target gene of miR-223. Gastric cancer cell line SGC7901 transfected with miR-223 or control oligonucleotide was resuspended in ECM gel and then was injected into the flank of nude mice, 4 weeks later, the nude mice were euthanized. The tumors were excised then were measured and weighted. SYBR-Green I-based real-time RT-PCR study was used to detect the level of miR-223 in 22 gastric cancer tissue and corresponding gastric mucosa tissues. Immunohistochemical method was applied to detect the protein of hFBXW7.
Gastric cancer cell line SGC7901, transfected with miR-223, showed significant reduction in cellular apoptosis and increased proliferation and invasion in vitro. Similar results were found in tumorigenesis assays performed in nude mice. Moreover, 19 of 22 cancer tissue samples highly expressed miR-223, when compared with patient-matched normal gastric mucosa. Specifically, patients with lymph node metastasis or metastatic disease (M1) at an advanced pathological stage showed significantly higher expression of miR-223. FBXW7/hCdc4 protein (FBW7) levels in gastric cancer cases were inversely correlated with miR-223 expression. Overexpression of miR-223 in gastric cancer cell lines decreased FBW7 expression at the translational level and decreased FBXW7/hCdc4-driven luciferase-reporter activity.
In summary, the data indicated that miR-223 targets FBXW7/hCdc4 expression at the post-transcriptional level and appears to regulate cellular apoptosis, proliferation, and invasion in gastric cancer. MiR-223 may serve as a novel therapeutic target in gastric cancer.
本研究旨在(a)确定 micro-223 (miR-223) 在胃癌中的作用，(b)阐明其对 FBXW7/hCdc4 基因的调控机制。
采用脂质体 2000 将人工 miR-223 和对照寡核苷酸转染至胃癌细胞系 SGC7901。通过流式细胞术分析 miR-223 组和对照组细胞的凋亡情况，通过 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四唑溴盐和集落形成实验检测细胞活力，检测 miR-223 组和对照组细胞的迁移情况；划痕愈合运动分析、Transwell 分析和 Western blot 实验检测 hFBXW7 的变化。荧光素酶报告实验通过将 pLUC-hFBXW7 WT-3'-UTR 与 pLMP-hsa-miR-223 或 pLMP 质粒（作为对照）共转染至 HEK293T 细胞中，用于检测 hFBXW7 是否为 miR-223 的直接靶基因。将 miR-223 或对照寡核苷酸转染的胃癌细胞系 SGC7901 悬浮在 ECM 凝胶中，然后注入裸鼠的侧腹，4 周后处死裸鼠。切除肿瘤并测量和称重。采用 SYBR-Green I 实时 RT-PCR 研究检测 22 例胃癌组织和相应胃黏膜组织中 miR-223 的水平。免疫组织化学方法检测 hFBXW7 蛋白。
转染 miR-223 的胃癌细胞系 SGC7901 ，体外细胞凋亡明显减少，增殖和侵袭能力增强。裸鼠肿瘤生成实验也得到了类似的结果。此外，与患者匹配的正常胃黏膜相比，22 例癌症组织样本中有 19 例高表达 miR-223。淋巴结转移或转移性疾病（M1）的患者在晚期病理阶段的 miR-223 表达明显升高。胃癌病例中 FBXW7/hCdc4 蛋白（FBW7）水平与 miR-223 表达呈负相关。胃癌细胞系中 miR-223 的过表达降低了 FBW7 在翻译水平的表达，并降低了 FBXW7/hCdc4 驱动的荧光素酶报告活性。
综上所述，数据表明 miR-223 靶向 FBXW7/hCdc4 的转录后表达，并可能调节胃癌中的细胞凋亡、增殖和侵袭。miR-223 可能成为胃癌的一种新的治疗靶点。
