基础医学与临床 ›› 2019, Vol. 39 ›› Issue (2): 175-181.

• 研究论文 • 上一篇    下一篇

人CYP4Z1基因重组慢病毒载体的构建、鉴定及抑制乳腺癌细胞增殖

余伟1,张秀1,王清1,唐立华1,吴杰2   

  1. 1. 宜昌市中心人民医院
    2. 三峡大学医学院
  • 收稿日期:2018-04-25 修回日期:2018-09-29 出版日期:2019-02-05 发布日期:2019-01-16
  • 通讯作者: 吴杰 E-mail:83182587@qq.com
  • 基金资助:
    CYP4Z1参与调节NMT活性在乳腺癌侵袭转移的作用及分子机制

Construction, identification of human CYP4Z1 gene recombinant lentiviral vector and its growth inhibition on breast cancer cells

  • Received:2018-04-25 Revised:2018-09-29 Online:2019-02-05 Published:2019-01-16
  • Contact: wu jiejie E-mail:83182587@qq.com
  • Supported by:
    The role of CYP4Z1 invovled in NMT activity on invasion and metastasis of breast cancer and its mechnisism

摘要: 目的 研究CYP4Z1 RNAi慢病毒表达载体作用人乳腺癌细胞后对CYP4Z1 mRNA表达及对细胞增殖和MAPK信号通路的影响。方法 用RT-PCR和Western blot检测MDA-MB-468、T-47D和MDA-MB-231细胞中CYP4Z1的表达。构建3种CYP4Z1 RNAi慢病毒载体,转染CYP4Z1高表达的乳腺癌细胞,筛选出干扰效果最好的靶点序列。用RT-PCR检测转染后CYP4Z1 mRNA表达。用MTT和Western blot分别检测CYP4Z1 siRNA后对乳腺癌细胞增殖和MAPK信号通路的影响。结果 在MDA-MB-468细胞中CYP4Z1 mRNA和蛋白表达显著高于MDA-MB-231及T-47D乳腺癌细胞(P<0.05)。成功构建了3组重组慢病毒载体(LV-CYP4Z1-RNAi-1/2/3),3组重组慢病毒感染MDA-MB-468细胞系后,CYP4Z1 mRNA的表达受到抑制,其中以LV-CYP4Z1-RNAi-2组抑制作用最显著,敲减率达到81%(P<0.01)。与正常对照组相比,CYP4Z1-RNAi-2组MDA-MB-468细胞增殖受到抑制,p-ERK1/2表达水平显著降低(P<0.05)。结论 成功构建了CYP4Z1 RNAi慢病毒表达载体,且CYP4Z1-RNAi-2可通过下调ERK1/2通路抑制乳腺癌MDA-MB-468细胞的增殖。

关键词: 关键词:CYP4Z1, MDA-MB-468, 乳腺癌, 慢病毒表达载体

Abstract: Objective To investigate the effects of CYP4Z1 RNAi lentiviral vector on CYP4Z1 mRNA expression, proliferation and MAPK signals of breast cancer cells. Methods The relative expression levels of CYP4Z1 in the MDA-MB-468, T-47D and MDA-MB-231 cells were detected by RT-PCR and Western blot. Three kinds of CYP4Z1 RNAi lentiviral vectors were constructed and infected into CYP4Z1 overexpression of breast cancer cells. Then, RT-PCR was used to screen out the CYP4Z1 RNAi lentiviral vector which could exert the best interference effect in breast cancer cells. The cells proliferation and the expression levels of MAPK signals of CYP4Z1 RNAi on breast cancer cells were detected by MTT methods and Western blot, respectively. Results The levels of CYP4Z1 mRNA and protein expression were highly in MDA-MB-468 cells than MDA-MB-231 and T-47D cells (P<0.05). Three groups of CYP4Z1 RNAi recombinant lentiviral vectors were successfully constructed (LV-CYP4Z1-RNAi-1/2/3). CYP4Z1 mRNA expression levels were decreased in MDA-MB-468 cells after infected with the three groups of recombinant lentiviral vectors, and the reduction rate of known-down CYP4Z1 in LV-CYP4Z1-RNAi-2 group reach to 81% (P<0.01). In addition, CYP4Z1-RNAi-2 significantly inhibited cells proliferation and the protein expression levels of p-ERK1/2 in MDA-MB-468 cells (P<0.05). Conclusions CYP4Z1 RNAi lentiviral vector is successfully constructed and CYP4Z1-RNAi-2 remarkably suppresses MDA-MB-468 cells proliferation via downregulation of ERK1/2 signal.

Key words: Key words: CYP4Z1, MDA-MB-468, breast cancer, lentiviral expression vector

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