人胃癌细胞BGC823中miR-200c靶基因产物波形蛋白的检测及功能研究

doi:10.16150/j.1671-2870.2020.04.017

摘要/Abstract

摘要：

目的： 采用RNA靶向干扰技术,研究人胃癌细胞BGC823中微小RNA（microRNA, miRNA, miR）-200c的靶点锌指E盒增强子结合蛋白2（zinc finger E-box enhancer binding protein 2, ZEB2）基因的表达情况,并检测相关的波形蛋白基因表达情况,观察miR-200c对胃癌BGC823细胞功能的影响。方法： 构建ZEB2基因表达的干扰质粒shZEB2,用Lipofectamine 2000转染胃癌BGC823细胞,用G418筛选稳定转染细胞株,进行实时定量聚合酶链反应（polymerase chain reaction,PCR）检测人BCG823细胞中ZEB2 mRNA、miR-200c及波形蛋白基因的表达水平,并用蛋白印迹法检测ZEB2蛋白、波形蛋白表达水平,噻唑蓝（methylthiazolyldiphenyl-tetrazolium bromide, MTT）法检测细胞增殖能力;Transwell小室检测细胞侵袭能力,划痕实验检测细胞迁移能力。结果： 干扰ZEB2分子表达后,miR-200c表达水平增高,而BGC823细胞的增殖、迁移及侵袭能力明显下降。结论： ZEB2是miR-200c的检测靶点,干扰ZEB2后,胃癌细胞BGC823的增殖、迁移及侵袭能力明显降低。

关键词: 胃癌, 锌指E盒增强子结合蛋白2, 波形蛋白

Abstract:

Objective: To investigate the expression of zinc finger E-box enhancer binding protein 2(ZEB2) gene in miR-200c in human gastric cancer BGC823 cells by RNA targeted interference technique, and detect expression of vimentin, so as to observe the effect of miR-200c on the function of gastric cancer BGC823 cells. Methods: The interference plasmids shZEB2 was constructed and transfected to BGC-823 cells in mediation of Lipofectamine 2000. The stable transfectants were screened by G418, and ZEB2 mRNA, miR-200c and vimentin was detected by real-time PCR in BCG823 cells, while protein expression levels of ZEB2 and vimentin by Western blotting. Proliferation of BGC-823 cells was evaluated by MTT assay. Transwell assay and wound healing assay were used to observe the invasion and migration ability of BCG823 cells, respectively. Results: With interfering ZEB2 expression, miR-200c expression level increased while proliferation, migration and invasion ability of BGC823 cells decreased significantly. Conclusions: ZEB2 may be the target of miR-200c, and the ability of BGC823 cells including proliferation, migration and invasion is significantly decreased after interference with ZEB2.

Key words: Gastric cancer, Zinc finger E-box enhancer binding protein 2 (ZEB2), Vimentin

中图分类号:

R735.2

杨翠萍, 杨晓金, 杨燕萍, 张梦茵, 谢玲, 俞骁珺, 蔡波尔, 陈登宇, 陈平, 吴云林. 人胃癌细胞BGC823中miR-200c靶基因产物波形蛋白的检测及功能研究[J]. 诊断学理论与实践, 2020, 19(04): 414-419.

YANG Cuiping, YANG Xiaojin, YANG Yanpin, ZHANG Mengyin, XIE Ling, YU Xiaojun, CAI Boer, CHEN Dengyu, CHEN Ping, WU Yunlin. Functional study and detection of vimentin produced by miR-200c target gene in human gastric cancer BGC823 cells[J]. Journal of Diagnostics Concepts & Practice, 2020, 19(04): 414-419.

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组别	1 d	2 d	3 d	4 d	5 d	6 d
Scramble	10 000	14 000	18 000	22 000	28 000	36 000
shZEB2	10 000	12 500	13 800	16 000	19 000	24 000