HAP1对乳腺癌细胞MCF-7生物学特性影响的观察
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摘要
目的:探讨亨廷顿相关蛋白1(HAP1)基因过表达对人乳腺癌细胞株MCF-7增殖、体外迁移侵袭和细胞凋亡的影响及其可能机制。方法:通过转染的方法将逆转录病毒pBabe-puro(嘌呤霉素)HAP1质粒和pBabe-puro质粒导入人乳腺癌细胞系MCF-7,用嘌呤霉素筛选稳定表达两质粒的细胞系,荧光定量PCR和蛋白质印迹法鉴定是否成功构建HAP1过表达细胞系;细胞增殖-毒性检测试剂盒(CCK-8)和克隆形成实验检测细胞的生长增殖,Transwell小室法检测细胞的侵袭和迁移,流式细胞仪检测细胞的凋亡。结果:成功构建稳定表达pBabe-HAP1的MCF-7-pBabe-puro-HAP1细胞模型。CCK-8检测72h细胞增殖率,MCF-7-pBabe-puro-HAP1为(75.97±6.76)%,明显低于MCF-7-pBabe-puro细胞(93.98±6.63)%(P=0.03)及MCF-7细胞(100.00±0.00)%,P=0.004;MCF-7-pBabe-puro-HAP1细胞克隆形成率为(22.67±1.26)%,明显低于MCF-7(35.00±0.50)%(P=0.000)和MCF-7-pBabe-puro细胞(33.83±0.76)%,P=0.000;Transwell小室侵袭和迁移实验表明,MCF-7-pBabe-puro-HAP1组的侵袭(3.33±0.58,P=0.000)和迁移(50.00±3.61,P<0.01)能力明显降低;流式细胞仪检测细胞凋亡,MCF-7-pBabe-puro-HAP1凋亡率为(8.03±0.15)%,高于MCF-7-pBabe-puro(3.13±0.25)%(P=0.000)和MCF-7细胞(3.33±0.35)%,P=0.000。结论:HAP1基因能够抑制肿瘤细胞增殖和迁移侵袭,并能诱导细胞凋亡,其可能作为一个抑癌基因在肿瘤发生发展中发挥重要作用。
OBJECTIVE:To investigate the effect of HAP1 gene overexpression on proliferation,migration and invasion capability,cell apoptosis of MCF-7 breast cancer cell line in vitro and possible mechanism.METHODS:Human breast cancer cell line MCF-7 was cultured and transfected with recombinant plasmid pBabe-puro-HAP1 or blank plasmid pBabe-puro.Real-time PCR and Western blot were used to detect the mRNA and protein expression of HAP1.The cell proliferation was detected by CCK-8 assay.The migration and invasion capability in vitro was detected by Transwell chamber assay.Clone formation assay was carried out to determine the clonogenicity.Cell apoptosis was determined by flow cytometry.RESULTS:The cell model MCF-7-pBabe-puro-HAP1 was successfully constructed,which was stably expressing pBabe-HAP1.After 72 h culturing,CCK-8 assay showed that the proliferation rate of MCF-7-pBabe-puro-HAP1 group was(75.97±6.76)%,which was higher than those of MCF-7-pBabe-puro(93.98±6.63)%(P=0.03) and MCF-7(100.00±0.00)%(P=0.004)groups.The abilities of migration(50.00±3.61,P<0.01) and invasion(3.33±0.58,P=0.000) of the MCF-7-pBabe-puro-HAP1 cells were obviously decreased as compared with those MCF-7 and MCF-7-pBabe-puro cells.The apoptosis rate of MCF-7-pBabe-puro-HAP1 cells was(8.03±0.15)%,which was also higher than those of MCF-7-pBabe-puro(3.13±0.25)%(P=0.000)and MCF-7(3.33±0.35)%(P=0.000) cells.The cloning efficiency of MCF-7-pBabe-puro-HAP1 was(22.67±1.26)%,which was lower than those of MCF-7(35.00±0.50)%(P=0.000) and MCF-7-pBabe-puro(33.83±0.76)%(P=0.000).CONCLUSIONS:HAP1 gene overexpression can inhibit the proliferation,migration and invasion capabilities,and induce the apoptosis of MCF-7 in vitro.It may play an important role in biological function as a tumor suppressor gene.
OBJECTIVE:To investigate the effect of HAP1 gene overexpression on proliferation,migration and invasion capability,cell apoptosis of MCF-7 breast cancer cell line in vitro and possible mechanism.METHODS:Human breast cancer cell line MCF-7 was cultured and transfected with recombinant plasmid pBabe-puro-HAP1 or blank plasmid pBabe-puro.Real-time PCR and Western blot were used to detect the mRNA and protein expression of HAP1.The cell proliferation was detected by CCK-8 assay.The migration and invasion capability in vitro was detected by Transwell chamber assay.Clone formation assay was carried out to determine the clonogenicity.Cell apoptosis was determined by flow cytometry.RESULTS:The cell model MCF-7-pBabe-puro-HAP1 was successfully constructed,which was stably expressing pBabe-HAP1.After 72 h culturing,CCK-8 assay showed that the proliferation rate of MCF-7-pBabe-puro-HAP1 group was(75.97±6.76)%,which was higher than those of MCF-7-pBabe-puro(93.98±6.63)%(P=0.03) and MCF-7(100.00±0.00)%(P=0.004)groups.The abilities of migration(50.00±3.61,P<0.01) and invasion(3.33±0.58,P=0.000) of the MCF-7-pBabe-puro-HAP1 cells were obviously decreased as compared with those MCF-7 and MCF-7-pBabe-puro cells.The apoptosis rate of MCF-7-pBabe-puro-HAP1 cells was(8.03±0.15)%,which was also higher than those of MCF-7-pBabe-puro(3.13±0.25)%(P=0.000)and MCF-7(3.33±0.35)%(P=0.000) cells.The cloning efficiency of MCF-7-pBabe-puro-HAP1 was(22.67±1.26)%,which was lower than those of MCF-7(35.00±0.50)%(P=0.000) and MCF-7-pBabe-puro(33.83±0.76)%(P=0.000).CONCLUSIONS:HAP1 gene overexpression can inhibit the proliferation,migration and invasion capabilities,and induce the apoptosis of MCF-7 in vitro.It may play an important role in biological function as a tumor suppressor gene.
引文
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[2]Sorensen SA,Fenger K.Causes of death in patients with Hun-tington’s disease and in unaffected first degree relatives[J].JMed Genet,1992,29(12):911-914.
[3]Liu DZ,Ander BP.Cell cycle inhibition without disruption of neuro-genesis Is a strategy for treatment of aberrant cell cycle disease:anupdate[J].The Scientific World Journal,2012,2012:491737.
[4]Hawley RG,Chen Y,Riz I,et al.An integrated bioinformaticsand computational biology approach identifies new BH3-Onlyprotein Candidates[J].Open Biol J,2012,5:6-16.
[5]St-Amand J,Yoshioka M,Tanaka K,et al.Transcriptome-wide i-dentification of preferentially expressed genes in the hypothala-mus and pituitary gland[J].Frond Endocrinol(Lausanne),2011,2:111.
[6]Martin EJ,Kim M,Velier J,et al.Analysis of Huntingtin-associ-ated protein 1in mouse brain and immortalized striatal neurons[J].J Comp Neurol,1999,403(4):421-430.
[7]Li SH,Li XJ.Huntingtin and its role in neuronal degeneration[J].Neuroscientist,2004,10(5):467-75.
[8]Wu LL,Zhou XF.Huntingtin associated protein 1and its func-tions[J].Cell Adh Migr,2009,3(1):71-76.
[9]Cape A,Chen X,Wang CE,et al.Loss of huntingtin-associatedprotein 1impairs insulin secretion from pancreaticβ-cells[J].CellMol Life Sci,2012,69(8):1305-1317.
[10]Liao M,Chen X,Han J,et al.Selective expression of huntingtin-associated protein 1inβ-Cells of the rat pancreatic islets[J].JHistochem Cytochem,2010,58(3):255-263.
[11]杨荣,杨友华,黄丹,等.多不饱和脂肪酸对阿尔茨海默病大鼠海马亨廷顿相关蛋白1表达的影响[J].中华行为医学与脑科学杂志,2010,19(10):882-884.
[12]韩金红,李和.两种亨廷顿相关蛋白1异构体-HAP1A和HAP1B在大鼠脊髓灰质内的分布特征[J].中国组织化学与细胞化学杂志,2010,19(3):211-215.
[13]姚玉霜,张婷婷,戚玉言,等.卵巢癌组织EGFR及下游信号分子的表达及其临床意义[J].中华肿瘤防治杂志,2011,18(3):212-215.
[14]Li SH,Yu ZX,Li CL,et al.Lack of huntingtin-associated pro-tein-1causes neuronal death resembling hypothalamic degenera-tion in huntington’s disease[J].J Nearosci,2003,23(17):6956-6964.