基础医学与临床 ›› 2016, Vol. 36 ›› Issue (2): 161-166.

• 研究论文 • 上一篇    下一篇

ESRP1剪接变异体的克隆及其对乳腺癌MDA-MB-231细胞增殖的影响

杨一平,吴玉君,赵雯文,李杉,乐灿,卢诗倩,袁成福   

  1. 三峡大学
  • 收稿日期:2015-06-29 修回日期:2015-11-10 出版日期:2016-02-05 发布日期:2016-01-21
  • 通讯作者: 袁成福 E-mail:yuancf46@ctgu.edu.cn
  • 基金资助:
    国家自科基金;湖北省自然科学基金;校人才启动基金;大学生科技创新项目

Cloning of ESRP1 splicing variants and its effects on MDA-MB-231 breast cancer cell line proliferation

  • Received:2015-06-29 Revised:2015-11-10 Online:2016-02-05 Published:2016-01-21
  • Contact: Cheng-Fu YUAN E-mail:yuancf46@ctgu.edu.cn

摘要: 目的 克隆ESRP1 剪接变异体并构建其慢病毒表达载体,研究其对乳腺癌细胞MDA-MB-231细胞增殖的影响。方法 以OVCAR3细胞的cDNA为模板,PCR扩增ESRP1剪接变异体,将其连接到带有Myc标签的pCMV-Myc真核表达载体;再以该真核表达载体为模板,将含有Myc标签的ESRP1剪接变异体克隆到慢病毒表达载体pLV-tTR/KRAB-Red上,从而构建带有Myc标签的ESRP1剪接变异体慢病毒表达载体;用该慢病毒表达载体转染293T细胞,包装慢病毒;用该慢病毒感染MDA-MB-231细胞,用MTT法检测该细胞增殖,用Western blot检测该细胞中PARP蛋白的表达。结果 成功构建带有Myc标签的ESRP1剪接变异体慢病毒表达载体,并包装出慢病毒;用该慢病毒感染MDA-MB-231细胞后,与空载体对照组比较,ESRP1过表达能明显抑制MDA-MB-231细胞增殖(P < 0.05); ESRP1过表达能诱导PARP蛋白的剪切。结论 成功构建带有Myc标签的ESRP1剪接变异体的慢病毒表达载体;ESRP1过表达能抑制MDA-MB-231细胞增殖,这可能与细胞死亡有关。

关键词: ESRP1, 乳腺癌, 细胞生长, 选择性剪接

Abstract: Objective To clone ESRP1 splicing variants and construct their lentivirus expression vector. To explore the effects of ESRP1 overexpression on MDA-MB-231 cell proliferation. Methods ESRP1 splicing variants were applified by PCR, in which the OVCAR3 cDNA was used as the template, and then the PCR products of ESRP1 splicing variants were ligated to Myc-tagged pCMV-Myc vector. ESRP1 splicing variants containing Myc-tag were cloned to lentivirus expression vector pLV-tTR/KRAB-Red. These lentivirus expression vectors containing Myc-tagged ESRP1 splicing variants were transfected into 293T cells, and lentivirus were made. MDA-MB-231 cells were infected with lentivirus, the cell proliferation was determined by MTT, and PARP cleavage was examined. Results The lentivirus expression vectors containing Myc-tagged ESRP1 splicing variants were constructed successfully, and the lentivirus were produced. ESRP1 overexpression could inhibit MDA-MB-231 cell proliferation and induce PARP cleavage in MDA-MB-231 cells. Conclusions The lentivirus expression vectors containing Myc-tagged ESRP1 splicing variants were constructed successfully. ESRP1 overexpression could inhibit MDA-MB-231 cell proliferation, and this may be correlated with cell death.

Key words: ESRP1, Breast cancer, Cell growth, Alternative splicing