miR-449a对人乳腺癌细胞MCF-7增殖及迁移能力的影响

doi:10.12047/j.cjap.5601.2017.121

中国应用生理学杂志 ›› 2017, Vol. 33 ›› Issue (6): 508-513.doi: 10.12047/j.cjap.5601.2017.121

miR-449a对人乳腺癌细胞MCF-7增殖及迁移能力的影响

王红磊, 肖奕, 武力, 马大昌

兰州大学第一医院乳腺科, 甘肃兰州 730000

收稿日期:2017-02-27 修回日期:2017-10-18 出版日期:2017-11-28 发布日期:2018-06-19
基金资助:
甘肃省自然科学基金项目（2011Y0163）；甘肃省自然科学基金项目（2012V0633）

Effects of miR-449a on proliferation and migration of human breast cancer cell line MCF-7

WANG Hong-lei, XIAO Yi, WU Li, MA Da-chang

Galactophore Department, the First Hospital of Lanzhou University, Lanzhou 730000, China

Received:2017-02-27 Revised:2017-10-18 Online:2017-11-28 Published:2018-06-19
Contact: 武力,Tel:15117273573;E-mail:wuligslz@163.com E-mail:wuligslz@163.com
Supported by:
甘肃省自然科学基金项目（2011Y0163）；甘肃省自然科学基金项目（2012V0633）

摘要/Abstract

摘要： 目的：通过敲低微小RNA （microRNA，miRNA）-449a的方法研究miR-449a对人乳腺癌细胞MCF-7的增殖和迁移能力的影响。方法：采用miRNA芯片在乳腺癌细胞MCF-7和人正常乳腺细胞MCF-10A筛选具有表达差异的miRNA；化学合成法制备miR-449a的抑制剂（inhibitor），转染后经real-time PCR验证表达的变化；细胞增殖CCK-8实验对转染后细胞增殖能力进行检测；划痕实验检测细胞转移能力，transwell小室实验检测细胞侵袭的改变；蛋白免疫印迹法（Western blot）实验对MCF-7细胞增殖和迁移相关的β-catenin和E-cadherin蛋白进行检测；通过生物信息学软件预测miR-449a潜在靶基因为Notch 1，荧光素酶实验检测Notch 1是miR-449a的靶基因。结果：分别收集MCF-7和MCF-10A细胞，芯片结果显示miR-449a在MCF-7细胞的表达水平显著高于MCF-10A；本研究将细胞分为未处理组（Mock组），阴性对照组（negative control组，NC组）和处理组，通过收集不同组MCF-7细胞进行试验，CCK-8结果显示miR-449a下调后MCF-7细胞增殖能力显著降低；划痕实验结果显示miR-449a表达降低导致MCF-7细胞转移能力降低；transwell实验结果显示MCF-7细胞侵袭受到抑制；Western blot结果发现miR-449a敲低后β-catenin表达降低，E-cadherin表达增加；荧光素酶试验结果显示，miR-449a能够显著降低Notch 1-3'-UTR质粒的荧光素活性（P<0.01）。结论：在乳腺癌细胞MCF-7中敲低miR-449a能够显著抑制癌细胞增殖和迁移，而这一变化可能通过降低Notch 1蛋白表达实现的。

关键词: miR-449a, 人乳腺癌MCF-7细胞, Notch1, 增殖, 迁移

Abstract: Objective: To study the effects of knockdown of miR-449a on the proliferation and migration of human breast cancer cell line Michigan Cancer Foundation-7 (MCF-7).Methods: Using miRNA chip screening of the differential expressions of miRNA in human breast cancer cell MCF-7 and normal breast cells MCF-10A. The inhibitor of miR-449a was synthesized by chemical and detected by real-time PCR after transfection aimed to verify the expression. Cell Counting Kit-8 (CCK-8) assay was used to detect the ability of cell proliferation after transfection with miR-449a inhibitor. Scratch assay was used to detect cell migration of MCF-7, and cell invasion ability was showed by transwell assay; The MCF-7 cell proliferation and migration related proteins, β-catenin and E-cadherin, were detected by Western blot. The potential target gene of miR-449a was predicted by bioinformatics software, and Notch homolog 1 (Notch 1) was proved to be the target gene of miR-449a by luciferase assay.Results: MCF-7 and MCF-10a cells were collected separately, and miRNA chip results showed that the level of miR-449a in MCF-7 cells was significantly higher than that of MCF-10A. In this study, the cells were divided into mock group, negative control group (NC group) and treatment group, the MCF-7 cells were collected before and after treatment and CCK-8 results showed that knockdown of miR-449a decreased MCF-7 cell proliferation ability significantly. Scratch assay results showed that downregulated miR-449a was related to the decreased metastasis of MCF-7 cells. Transwell results showed that knockdown of miR-449a inhibited the invasion of MCF-7 cells. Western blot showed the expression of β-catenin was decreased and the expression of E-cadherin was increased after knockdown of miR-449a. Luciferase assay showed that miR-449a could significantly decrease the luciferase activity of Notch homolog 1-untranslated region (Notch 1-3'-UTR) plasmid (P<0.01).Conclusion: Inhibition of miR-449a in breast cancer cell line MCF-7 can significantly inhibit the proliferation and migration of cancer cells, which may be achieved by decreasing the expression of Notch 1 protein.

Key words: miR-449a, human breast cancer cell line MCF-7, proliferation, migration

王红磊, 肖奕, 武力, 马大昌. miR-449a对人乳腺癌细胞MCF-7增殖及迁移能力的影响[J]. 中国应用生理学杂志, 2017, 33(6): 508-513.

WANG Hong-lei, XIAO Yi, WU Li, MA Da-chang. Effects of miR-449a on proliferation and migration of human breast cancer cell line MCF-7[J]. CJAP, 2017, 33(6): 508-513.

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miR-449a对人乳腺癌细胞MCF-7增殖及迁移能力的影响

Effects of miR-449a on proliferation and migration of human breast cancer cell line MCF-7

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