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《天津医科大学学报》[ISSN:1006-8147/CN:12-1259/R]

卷:

20卷

期数:

2014年05期

页码:

337-341

栏目:

基础医学

出版日期:

2014-09-20

文章信息/Info

Title:

 Construction of HOXA5 eukaryotic expression plasmid of and its biological significance   in breast cancer cells

文章编号:

1006-8147(2014)05-0337-05

作者:

伦淑敏

（天津医科大学肿瘤医院肿瘤研究所，.国家肿瘤临床医学研究中心，天津市肿瘤防治重点实验室 ，天津，300060）

Author(s):

LUN Shu-min

(Cancer Institute and Hospital，Tianjin Medical University ，National Clinical Research Center of Cancer,  Key Laboratory of Cancer Prevention and Therapy.Tianjin 300060 ，China)

关键词:

HOXA5 ; 乳腺癌;  质粒 ; 迁移

Keywords:

HOXA5;  breast cancer;  plasmid;  migration

分类号:

R73

DOI:

-

文献标志码:

A

摘要:

目的：构建HOXA5基因真核表达重组质粒，并转染乳腺癌细胞株观察其对细胞迁移能力的影响。方法：实时定量聚合酶链反应（RT-QPCR）检测MCF7、BT474及MDA-MB-231等3种乳腺癌细胞中HOXA5 mRNA的表达。采用全基因合成法合成HOXA5编码区序列，克隆入pcDNA3.1（+）HA载体，构建重组质粒pcDNA3.1-HOXA5。重组质粒转染乳腺癌细胞，采用Western blot及RT-QPCR检测HOXA5的表达效率。观察细胞形态并通过细胞划痕实验检测细胞的迁移能力。结果：3株乳腺癌细胞中，MDA-MB-231细胞中HOXA5 mRNA表达水平较低。其基因测序结果显示与预期片段大小一致，表明HOXA5重组质粒构建成功。HOXA5重组质粒瞬时转染MDA-MB-231细胞后，到 mRNA和蛋白表达水平明显上调MDA-MB-231细胞的迁移能力EMT相关转录因子Twist的表达下调。结论：成功构建HOXA5过表达重组质粒，在MDA-MB-231中过表达HOXA5抑制乳腺癌的迁移能力。

Abstract:

Objective: To construct eukaryotic expression plasmid of pcDNA3.1-HOXA5 and examine its effect on the migration of the breast cancer cell line. Methods:The HOXA5 mRNA levels were detected in MCF7,BT474 and MDA-MB-231 cells, respectively, by real-time quantitative PCR. The full-length coding sequence of HOXA5 was amplified using standard RT-QPCR. The amplified HOXA5 was cloned into pcDA3.1 vector. And the eukaryotic expression plasmid pcDNA3.1-HOXA5 was transfected into breast cancer cells. RT-QPCR and Western Blot were performed to measure the expression level of HOXA5. The cell morphology changes were observed. The migration ability of the cells was also observed through scratch assay. Results: The HOXA5 mRNA expression level of MDA-MB-231 was low in MDA-MB-231 cells. Western Blot and RT-QPCR indicated that transfection of HOXA5 expression vector into MDA-MB-231 cells resulted in enhanced mRNA/protein levels of HOXA5. The forced HOXA5 expression significantly decreased cell migration of MDA-MB-231 and down-regulated the expression of Twist. Conclusion: The eukaryotic expression plasmid of pcDNA3.1-HOXA5 can be successfully constructed. Over-expression of HOXA5 inhibits the migration ability of MDA-MB-231cells.

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备注/Memo

备注/Memo:

作者简介 伦淑敏（1988-），女，硕士在读，研究方向：乳腺癌相关基因的转录调控机制； E-mail：lunshuminvs@163.com。

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更新日期/Last Update: 2014-09-25