天津医药

• 细胞与分子生物学 •    下一篇

ZEB2基因 3′UTR区转染对人胃黏膜上皮细胞GES-1 增殖、侵袭、迁移的影响*

李素丽1,周芳1,张庆瑜2,贾文亮1,张安玲1   

  1. 1. 天津市天津医科大学总医院消化内科
    2. 天津医科大学总医院消化内科
  • 收稿日期:2014-01-24 修回日期:2014-02-19 出版日期:2014-05-15 发布日期:2014-05-15
  • 通讯作者: 张庆瑜

Effects of ZEB2 3′UTR Transfection on Proliferation ,Invasion and Migration of Human Gastric Epithelial Cell GES-1

  • Received:2014-01-24 Revised:2014-02-19 Published:2014-05-15 Online:2014-05-15
  • Contact: 苏娟 张庆瑜 康春生

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摘要：

【摘要】目的探讨E盒结合锌指蛋白（ZEB）2基因3′非翻译区（3′UTR）转染人胃黏膜上皮细胞GES-1后对其增殖、侵袭、迁移的影响。方法将人工合成的ZEB23′UTR质粒及miR-200b micmics采用脂质体Lipofectamine2000转染GES-1细胞。分别设对照组、突变组和ZEB23′UTR组，qRT-PCR检测转染后miR-200a/b/c及ZEB1/ZEB2mRNA的表达。再设对照组、ZEB23′UTR组、ZEB23′UTR+无义序列组和ZEB23′UTR+miR-200b micmics组。Western blot检测转染后ZEB1/ZEB2、基质金属蛋白酶（MMP）-2/9、增殖细胞核抗原（PCNA）蛋白的表达；Transwell侵袭实验和划痕实验检测细胞侵袭迁移能力；MTT法检测细胞增殖活性。结果与对照组及突变组相比，转染ZEB23′UTR组miR-200a/b/c的表达均下调，以miR-200b最为明显，ZEB1/ZEB2mRNA及蛋白水平表达上调，其细胞迁移、侵袭、增殖活性均增强（P<0.05）。ZEB23′UTR+miR-200b micmics组较ZEB23′UTR组迁移、侵袭、增殖活性降低。结论 ZEB23′UTR可能通过调控miR-200a/b/c的表达进而影响其对靶基因的转录后调控，增加细胞的侵袭、迁移能力，导致GES-1细胞的恶性转化倾向。

关键词: 基因, 同源盒, 3′非翻译区, 微RNAs, 胃黏膜, 上皮细胞, 肿瘤转移, E盒结合锌指蛋白

Abstract:

[Abstract] Objective To explore the effects of zinc finger E-box binding protein (ZEB)23′UTR gene transfection
on proliferation, invasion and migration in human gastric epithelial cell line GES-1. Methods The synthetic ZEB23′UTR
and miR-200b micmics were transfected into GES-1cell line by lipofectamine2000. We set up control grop, the mutation group and ZEB23′UTR group. Real-time quantitative PCR was performed to evaluate the expression levels of miR-200a/b/c and ZEB1/ZEB2mRNAs after transfection．And then we set up control group, ZEB23′UTR group, ZEB23′UTR+negative control group and ZEB23′UTR+miR-200b micmics group. The protein expression levels of ZEB1, ZEB2, matrix metallopro?teinases (MMP) 2/9and proliferating cell nuclear antigen (PCNA) were detected by Western blot assay. The invasion and migration capability were analyzed by transwell assay and wound healing test. MTT assay was used to detect the proliferation ability.Results Compared with control group and mutation group, the expressions of miR-200a/b/c were significantly decreased, especially for miR-200b. And the expressions of ZEB1/ZEB2were significantly increased at both mRNA and protein levels after transfected with the ZEB23′ UTR, enhancing the capability of migration，invasion，and proliferation (P<0.05). Compared with ZEB23′UTR group, the capabilities of proliferation，invasion and migration were significantly lower in combined group.Conclusion ZEB23′ UTR can increase the ability of cell proliferation, invasion and metastasis through regulating the levels of miR-200a/b/c, and then influence the regulation of transcription of the target gene, which could lead to malignant transformation of GES-1cells.

Key words: genes, homeobox, 3′untranslated regions, microRNAs, gastric mucosa, epithelial cells, neoplasm metastasis, E盒结合锌指蛋白