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ZEB2 3′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ZEB2 3′UTR组,qRT-PCR检测转染后miR-200a/b/c及ZEB1/ZEB2mRNA的表达。再设对照组、ZEB2 3′UTR组、ZEB2 3′UTR+无义序列组和ZEB2 3′UTR+miR-200b micmics组。West-ern 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ZEB2 3′UTR组miR-200a/b/c的表达均下调,以miR-200b最为明显,ZEB1/ZEB2 mRNA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)。ZEB2 3′UTR+miR-200b micmics组较ZEB2 3′UTR组迁移、侵袭、增殖活性降低。结论ZEB2 3′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。
Objective To explore the effects of zinc finger E-box binding protein （ZEB）2 3′UTR gene transfection on proliferation, invasion and migration in human gastric epithelial cell line GES-1. Methods The synthetic ZEB2 3′UTR and miR-200b micmics were transfected into GES-1 cell line by lipofectamine 2000. We set up control grop, the mutation group and ZEB2 3′UTR group. Real-time quantitative PCR was performed to evaluate the expression levels of miR-200a/b/c and ZEB1/ZEB2 mRNAs after transfection.And then we set up control group, ZEB2 3′UTR group, ZEB2 3′UTR＋negative control group and ZEB2 3′UTR＋miR-200b micmics group. The protein expression levels of ZEB1, ZEB2, matrix metallopro-teinases （MMP） 2/9 and proliferating cell nuclear antigen （PCNA） were detected by Western blot assay. The invasion and mi-gration 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 de-creased, especially for miR-200b. And the expressions of ZEB1/ZEB2 were significantly increased at both mRNA and pro-tein levels after transfected with the ZEB2 3′UTR, enhancing the capability of migration,invasion,and proliferation （P ＆lt;0.05）. Compared with ZEB2 3′UTR group, the capabilities of proliferation,invasion and migration were significantly lower in combined group. Conclusion ZEB2 3′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-1 cells.
出处
《天津医药》
CAS
北大核心
2014年第5期401-405,I0001,共6页
Tianjin Medical Journal
基金
国家自然科学基金资助项目(项目编号:81172356)
天津市自然科学基金重点项目(项目编号:10JCZDJC18500)
关键词
3′非翻译区
转染
胃黏膜
上皮细胞
细胞增殖
肿瘤转移
E盒结合锌指蛋白
3′untranslated regions
microRNAs
transfection
gastric mucosa
epithelial cells
cell proliferation
neoplasm metastasis
zinc finger E-box binding homeobox
