PinX1基因真核表达载体的构建及其对乳腺癌MCF-7细胞增殖的抑制作用

doi:10.3969/j.issn.1007-2861.2013.06.015

上海大学学报(自然科学版) ›› 2013, Vol. 19 ›› Issue (6): 631-635.doi: 10.3969/j.issn.1007-2861.2013.06.015

PinX1基因真核表达载体的构建及其对乳腺癌MCF-7细胞增殖的抑制作用

胡宗1, 梁桑华2, 马文丽1,2, 郑文岭1,3

1. 上海大学电子生物研究所, 上海200444; 2. 南方医科大学基因工程研究所, 广州510515; 3. 华南基因组研究中心, 广州510800

出版日期:2013-12-30 发布日期:2013-12-30
通讯作者: 马文丽(1964—), 女, 教授, 博士生导师, 研究方向为基因诊断与基因治疗. E-mail:gendustry@gmail. com
作者简介:马文丽(1964—), 女, 教授, 博士生导师, 研究方向为基因诊断与基因治疗.
基金资助:
国家自然科学基金资助项目(10972130)

Construction of Recombinant Eukaryotic Expression Vector for PinX1 and Its Roles in Inhibiting Proliferation of Human Breast Cancer MCF-7 Cell Line

HU Zong1, LIANG Sang-hua2, MA Wen-li1,2, ZHENG Wen-ling1,3

1. Bio-electronic Center, Shanghai University, Shanghai 200444, China; 2. Institute of Genetic Engineering, South Medical University, Guangzhou 510515, China; 3. South China Genomics Research Center, Guangzhou 510800, China

Online:2013-12-30 Published:2013-12-30

摘要/Abstract

摘要： 探讨了PinX1 基因在乳腺癌MCF-7 细胞生长和细胞周期中的作用, 初步探讨了该基因用于乳腺癌临床治疗的可行性. 采用RT-PCR 技术从293-T 细胞中扩增PinX1 基因, 将其克隆入真核表达载体pEGFP-C1 中, 再将重组质粒转染MCF-7 细胞. 通过real-time PCR 检测PinX1 基因的mRNA 表达, 用MTT 法检测转染前后细胞生长曲线的变化, 用流式细胞仪检测转染目的基因后细胞生长周期的改变. 检测结果表明, PinX1 基因已经在转染后MCF-7 细胞的细胞核内稳定表达, 乳腺癌细胞生长明显减缓(P <0.05), 增殖变慢(P <0.05), 细胞生长阻滞于G0/G1 期, 说明PinX1 基因可抑制乳腺癌MCF-7 细胞的生长和增殖.

关键词: PinX1基因, 乳腺癌, 细胞增殖, 载体构建

Abstract: This paper explores the effects of PinX1 gene on the growth and the cell cycles of breast carcinoma cell line MCF-7 and the potential in treatment of breast cancer. The PinX1 mRNA was amplified from 293-T cell with RT-PCR and inserted into pEGFP-C1 vector. The recombined eukaryotic expression vector of PinX1 was confirmed by enzyme digestion, and transfected into the breast carcinoma cell line MCF-7 using a liposome method. The expressed mRNA was detected by real-time PCR, and the cell growth and cell cycles by MTT and flow cytometry, respectively. Results showed that PinX1 transfection into the breast cancer MCF-7 cell line was stable and the transfected gene was integrated into nucleolus DNA of transfected cells. Transfection of PinX1 gene could significantly inhibit the growth and proliferation of breast cancer cells (P < 0.05) and the cell growth was arrested at G0/G1 stage. The conclusion is that PinX1 gene could inhibit growth and proliferation of breast cancer cells.

Key words: PinX1 gene, breast cancer, cell proliferation, vector construction

中图分类号:

R 736.3

胡宗1, 梁桑华2, 马文丽1,2, 郑文岭1,3. PinX1基因真核表达载体的构建及其对乳腺癌MCF-7细胞增殖的抑制作用[J]. 上海大学学报(自然科学版), 2013, 19(6): 631-635.

HU Zong1, LIANG Sang-hua2, MA Wen-li1,2, ZHENG Wen-ling1,3. Construction of Recombinant Eukaryotic Expression Vector for PinX1 and Its Roles in Inhibiting Proliferation of Human Breast Cancer MCF-7 Cell Line[J]. Journal of Shanghai University（Natural Science Edition）, 2013, 19(6): 631-635.

参考文献

[1] Blasco M A. Telomeres and human disease: ageing, cancer and beyond [J]. Nat Rev Genet, 2005, 6(8): 611-622.

[2] Zhou X Z, Lu K P. The Pin2 /TRF1 interacting protein PinX1 is a potent telomerase inhibitor [J]. Cell, 2001, 107 (3): 347-359.

[3] Liao C, Zhao M J, Song H, et al. Identification of the gene for a novel liver-related putative tumor suppressor at a high frequency loss of heterozygosity region of chromosome 8p23 in human hepatocellular carcinoma [J]. Hepatology, 2000, 10: 721-722.

[4] 孙文旦. PinX1 基因真核表达载体的构建及其在Hek293 细胞中的表达[J]. 临床检验, 2009, 27: 445-447.

[5] Won S P, Jong H L, Jik Y P, et al. Genetic analysis of the liver putative tumor suppressor gene in hepatocelluar carcinomas [J]. Cancer Let, 2002, 178(2): 199-207.

[6] de Lange T. Shelterin: the protein complex that shapes and safeguards human telomeres [J]. Genes Dev, 2005, 19(18): 2100-2110.

[7] van Steensel B, De Lange T. Control of telomere length by the human telomeric protein TRF1 [J]. Nature, 1997, 385: 740-743.

[8] Chong L, Van S B, Broccoli D, et al. A human telomeric protein [J]. Science, 1995, 270(5242): 1663-1667.

[9] Ancelin K, Brunori M, Bauwens S, et al. Targeting assay to study the cis functions of human telomeric proteins: evidence for inhibition of telomerase by TRF1 and for activation of telomere degradation by TRF2 [J]. Mol Cell Biol, 2002, 22(10): 3474-3487.

[10] Soohoo C Y, Shi R, Lee T H, et al. Telomerase inhibitor PinX1 provides a link between TRF1 and telomerase to prevent telomere elongation [J]. J Biol Chem, 2011, 286: 3894-3906.

[11] Zhou X Z, Huang P, Shi R, et al. The telomerase inhibitor PinX1 is a major haploinsufficient tumor suppressor essential for chromosome stability [J]. J Clin Invest, 2011, 121: 1266-1282.

[12] Chen G, Da L, Wang H, et al. HIV-Tat-mediated delivery of an LPTS functional fragment inhibits telomerase activity and tumorigenicity of hepatoma cells [J]. Gastroenterology, 2011, 140(1): 332-343.

[13] Zhang L, Jiang Y, Zheng Y, et al. Selective killing of Burkitt’s lymphoma cells by mBAFF-targeted delivery of PinX1 [J]. Leukemia, 2011, 25(2): 331-340.

[14] Wang H B, Wang W Q, Wang X W, et al. PinX1 gene transfection enhances the sensitivity of gastric carcinoma cell line to 5-fluorouracil [J]. Hepatogastroenterology, 2011, 58(106): 682-686.

[15] Harley C B. Telomerase and cancer therapeutics [J]. Nat Rev Cancer, 2008, 8(3): 167-179.

[16] Joseph I, Tressler R, Bassett E, et al. The telomerase inhibitor imetelstat depletes cancer stem cells in breast and pancreatic cancer cell lines [J]. Cancer Res, 2010, 70(22): 9494-9504.

PinX1基因真核表达载体的构建及其对乳腺癌MCF-7细胞增殖的抑制作用

Construction of Recombinant Eukaryotic Expression Vector for PinX1 and Its Roles in Inhibiting Proliferation of Human Breast Cancer MCF-7 Cell Line

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 4

编辑推荐

Metrics

本文评价

[1]	苑婕, 马方玮, 李梦云, 曹庆, 郑伟尉, 万嗣宝. 水蜜桃E3泛素连接酶PpARI1基因的克隆及表达载体构建[J]. 上海大学学报(自然科学版), 2019, 25(4): 604-611.
[2]	苏杭, 王保垒, 李华顺, 朱晨光. Numb基因不同亚型对乳腺癌细胞MCF-7增殖和迁移能力的影响[J]. 上海大学学报(自然科学版), 2018, 24(3): 476-485.
[3]	朱浩1, 丁胜光1, 黄海涛1, 许嘉鸿2, 仲崇俊1. 新生大鼠心脏再生模型的改良及评价模式[J]. 上海大学学报(自然科学版), 2017, 23(3): 387-394.
[4]	康佳, 雷炳莉, 刘倩, 王学彤, 吴明红, 徐刚, 傅家谟. 上海河流沉积物的有机提取物对3 种细胞的毒性效应[J]. 上海大学学报(自然科学版), 2013, 19(4): 393-399.