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

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ZEB2mRNA、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, andZEB2mRNA, miR-200c and vimentin was detected by real-time PCR in BCG823 cells, while protein expression levels ofZEB2and 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 interferingZEB2expression, miR-200c expression level increased while proliferation, migration and invasion ability of BGC823 cells decreased significantly.Conclusions:ZEB2may be the target of miR-200c, and the ability of BGC823 cells including proliferation, migration and invasion is significantly decreased after interference withZEB2.

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.

图/表6

图1

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

表1

图2

图3

图4

图5

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