转移相关1基因对胃癌细胞生物学行为的影响  

Effects of MTA1 on biological behaviors of gastric cancer cells

作　　者：王劲松 钱海利 王海娟 许东奎 Wang Jinsong;Qian Haili;Wang Haijuan;Xu Dongkui(State Key Laboratory of Molecular Oncology,National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital,Chinese Academy of Medical Sciences and Peking Union Medical College,Beijing 100021,China;Department of VIP,National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital,Chinese Academy of Medical Sciences and Peking Union Medical College,Beijing 100021,China)

机构地区：[1]国家癌症中心 国家肿瘤临床医学研究中心 中国医学科学院 北京协和医学院肿瘤医院分子肿瘤学国家重点实验室,100021 [2]国家癌症中心 国家肿瘤临床医学研究中心 中国医学科学院 北京协和医学院肿瘤医院VIP病房,100021

出　　处：《中华肿瘤杂志》2018年第8期580-586,共7页Chinese Journal of Oncology

基　　金：中国医学科学院医学与健康创新科技工程项目（2016-12M-1,001）;国家基础研究计划973项目（2015CB553904）;国家自然科学基金（81372158、81372159、81572842、81672459）;分子肿瘤学国家重点实验室开放课题（SKL-KF-2017-16）;分子肿瘤学国家重点实验室自主创新课题（SKL-2017-16）

摘　　要：目的研究肿瘤转移相关1（MTA1）基因对胃癌细胞迁移、侵袭、生长等多种生物学特性的影响。方法利用pSilencer3.1－MTA1－siRNA载体构建稳定敲降MTA1的人胃癌BGC－823细胞，通过Boyden实验、划痕愈合实验、克隆形成实验和四甲基偶氮唑蓝（MTT）法检测敲降MTA1基因对BGC－823细胞多种生物学行为的影响。同时，利用pcDNA3－MTA1质粒过表达MTA1基因进行反向验证。在裸鼠皮下分别注射pSilencer3.1－MTA1－siRNA转染细胞、pcDNA3－MTA1转染细胞和对照细胞，检测MTA1基因对胃癌BGC823细胞体内成瘤能力的影响。采用逆转录聚合酶链反应（RT－PCR）测定pSilencer3.1－MTA1－siRNA转染组、pcDNA3－MTA1转染组和对照组细胞中integrinβ1、cyclinD1和尿激酶受体（uPAR）等转移相关基因的表达，观察其与MTA1表达的相关性。结果成功构建了稳定敲降及过表达MTA1的BGC－823细胞。Boyden实验和划痕愈合实验结果显示，与对照组细胞比较，pSilencer3.1－MTA1－siRNA转染组发生迁移和侵袭的细胞数明显减少[分别为（25±2）和（12±1）个]，pcDNA3－MTA1转染组发生迁移和侵袭的细胞数则明显增多[分别为（218±2）和（269±3）个]，差异均有统计学意义（均P〈0.01）。MTT法和克隆形成实验结果表明，pcDNA3－MTA1转染组细胞生长加快，克隆形成能力增强，pSilencer3.1－MTA1－siRNA转染组细胞与之相反。皮下成瘤实验显示，pSilencer3.1－MTA1－siRNA转染组肿瘤体积和重量分别为（711.32±284.30）mm3和（0.87±0.24）g，明显小于对照组[（1 482.41±511.90）mm3和（1.39±0.30）g]，而pcDNA3－MTA1转染组肿瘤体积和肿瘤重量分别为（3 158.73±1 823.22）mm3和（2.23±0.51）g，明显大于对照组，差异均有统计学意义（均P〈0.05）。RT－PCR检测结果显示，与对照组细胞比较，pSilencer3.1－MTA1－siRNA转染组细胞cyclinD1、integrinβ1和uPAR mRNA表达下调，而pcDNA3－MTAObjectiveTo study the effects of metastasis associated 1 （MTA1） on biological characteristics such as migration, invasion and proliferation of gastric cancer （GC） cells.MethodspSilencer3.1-MTA1-siRNA vector was used to establish human gastric cancer BGC-823 cell lines with constitutive MTA1-knockdown. Boyden, wound healing, clony forming assay and 3-（4, 5-dimethyl-2-thiazolyl）-2, 5-diphenyl-2H tetrazolium bromide （MTT） assay were performed to identify the effects of MTA1-deficiency on the biological behaviors of BGC-823 cells in vitro. Simultaneously, MTA1 overexpressed BGC-823 cell line was established by pcDNA3-MTA1 plasmid transfection for reverse verification. In addition, the role of MTA1 in the tumorigenicity of gastric cancer BGC823 cells in vivo was examined by subcutaneous injection of BGC-823 cells expressing different MTA1 levels into nude mice. Reverse transcription-polymerase chain reaction （RT-PCR） and western blot were used to detect the expression levels of integrin β1, cyclin D1 and uPAR in pSilencer3.1-MTA1-siRNA, pcDNA3-MTA1 transfected cells and control cells.ResultsMTA1 knocked down or upregulated BGC-823 cell lines were successfully generated by transfecting pSilencer3.1-MTA1-siRNA or pcDNA3-MTA1 vector with lipofectamine 2000, respectively. The Boyden and wound healing experiments showed metastasis and invasion ability in MTA1 knocked down cells （25±2, 12±1） were significantly decreased when compared with those of control （78±2, 50±2） and MTA1-overexpressed groups （218±2, 269±3; P〈0.05）. The results of MTT assay and colony forming assay were significantly decreased when compared with those of showed that MTA1 overexpressed cells grew more rapidly and formed more colonies in vitro and induced worse malignant tumors in vivo, while MTA1 knocked down cells presented the reversed phenotype[control group （1 482.41±511.90） mm3, （1.39±0.29）g; MTA1 overexpressed group [（3 158.73±1 823.22） mm3, （2.23±0.51）g; MTA1-downregulated group （711.

关 键 词：胃肿瘤 转移相关基因1 生物学行为 细胞系 裸鼠 

分 类 号：R735.2[医药卫生—肿瘤]