MicroRNA-223 functions as an oncogene in human gastric cancer by targeting FBXW7/hCdc4
MicroRNA-223 functions as an oncogene in human gastric cancer by targeting FBXW7/hCdc4
复制标题
MicroRNA-223 通过靶向 FBXW7/hCdc4 作为人类胃癌的癌基因
DOI：
10.1007/s00432-012-1154-x
复制
发表时间：
2012-05-01
影响因子：
3.6
通讯作者：
Wu, Wenxi
中科院分区：
文献类型：
--
作者：
Li, Jinhai;Guo, Yuanyuan;Wu, Wenxi
目的本研究的目的是(a)确定微-223 （miR-223）在胃癌中的作用，(b)阐明其对FBXW7/hCdc4基因的调控机制。材料与方法采用Lipofectamine2000将人工合成的miR-223及对照寡核苷酸转染胃癌细胞株SGC7901。采用流式细胞术分析miR-223组和对照组细胞的凋亡情况，采用3-（4,5-二甲基噻唑-2-基）-2,5-二苯基溴化四唑和集落形成法检测细胞活力，观察miR-223组和对照组细胞的迁移情况；采用抓伤愈合动力法、Transwell法和Western blot法测定hFBXW7的方差。荧光素酶报告基因检测，将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是否是miR-223的直接靶基因。转染miR-223或对照寡核苷酸的胃癌细胞株SGC7901用ECM凝胶重悬，注射于裸鼠侧腹，4周后处死裸鼠。切除肿瘤后测量并称重。采用基于SYBR-Green i的实时RT-PCR方法检测22例胃癌组织及相应胃黏膜组织中miR-223的水平。采用免疫组化法检测hFBXW7蛋白。结果转染miR-223后，胃癌细胞株SGC7901的细胞凋亡明显减少，增殖和侵袭能力增强。在裸鼠的肿瘤发生实验中也发现了类似的结果。此外，与患者匹配的正常胃粘膜相比，22个癌组织样本中有19个高表达miR-223。具体而言，淋巴结转移或转移性疾病（M1）患者在病理阶段晚期miR-223的表达明显升高。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的过表达在翻译水平上降低了FBW7的表达，并降低了FBXW7/ hcdc4驱动的荧光素酶报告蛋白活性。综上所述，这些数据表明miR-223在转录后水平上靶向FBXW7/hCdc4的表达，并可能调节胃癌细胞的凋亡、增殖和侵袭。MiR-223可能成为胃癌新的治疗靶点。
AimsThe 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.Materials and methodsArtificial 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.ResultsGastric 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.ConclusionIn 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.