天津医药

天津医药 ›› 2019, Vol. 47 ›› Issue (5): 463-467.doi: 10.11958/20181959

• 细胞与分子生物学 • 上一篇    下一篇

慢病毒介导CK18基因沉默对人乳腺癌BT549细胞增殖、凋亡和侵袭能力的影响

张青 1，黄钱 1，张春燕 1，刘晓燕 2，曾凡才 1，甘淋 1△   

  1. 基金项目：四川省科技厅应用基础研究计划项目（14JC0155）；泸州-西南医大联合项目（2017LZXNYD-J11) 作者单位：1西南医科大学生物化学与分子生物学教研室（邮编646000），2医学基础研究中心 作者简介：张青（1988），女，硕士在读，主要从从事乳腺癌分子标志物的研究 △通讯作者 E-mail: gl-gump@163.com
  • 收稿日期:2018-12-07 修回日期:2019-02-27 出版日期:2019-05-15 发布日期:2019-05-15
  • 通讯作者: 甘淋 E-mail:gl-gump@163.com
  • 基金资助:
    四川省科技厅应用基础研究计划项目

Effects of lentiviral vector-mediated CK18 gene on cell proliferation, apoptosis and metastasis in breast cancer cell line BT549

ZHANG Qing1 , HUANG Qian1 , ZHANG Chun-yan1, LIU Xiao-yan 2, ZENG Fan-cai1 , GAN Lin1△   

  1. 1 Department of Biochemistry and Molecular Biology, 2 The Research Center for Preclinical Medicine, Southwest Medical University, Luzhou 646000, China △Corresponding Author E-mail: gl-gump@163.com
  • Received:2018-12-07 Revised:2019-02-27 Published:2019-05-15 Online:2019-05-15

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摘要： 摘要：目的 检测靶向沉默细胞角蛋白18（CK18）基因对人乳腺癌BT549细胞增殖、凋亡、侵袭运动的影响，并探 讨其潜在的分子机制。方法 建立 CK18 稳定沉默的 BT549 细胞株，分为 3 组，以 CK18（CK18-sh21、CK18-sh22、 CK18-sh23及CK18-sh24）靶向沉默的细胞为实验组CK18-shRNA，加入空载体的为阴性对照（sh-Con）组，未处理为 空白对照（Wt）组。采用蛋白印迹法（Western blot）对细胞系进行CK18表达鉴定。采用CCK-8法、平板克隆形成法检 测 CK18 表达缺失对细胞增殖能力的影响；通过流式细胞技术（PE–Annexin V 双染法）检测 CK18 基因沉默后对 BT549细胞凋亡的影响；通过PI-FACS 法检测沉默CK18对BT549细胞周期的影响；采用划痕试验、Transwell法检测 CK18沉默对细胞运动及侵袭能力的影响；采用Western blot检测与侵袭转移相关的分子E-钙黏蛋白（E-cadherin）和 波形蛋白（vimentin）表达情况。结果 建立了CK18沉默的BT549细胞系，CK18的表达被显著抑制，CK18-sh23组相 对sh-Con组抑制率最高，可达73%。与Wt组、sh-Con组相比，CK18-shRNA细胞增殖能力在24 h、48 h和72 h降低， 克隆形成能力下降，细胞运动、侵袭能力下降，细胞凋亡率和G2期细胞比例均增加（P＜0.05）。与sh-Con组和Wt组 比较，CK18表达降低能促进E-cadherin表达，抑制vimentin表达（P＜0.05）。结论 CK18基因沉默能有效抑制人乳 腺癌BT549细胞的增殖、运动、侵袭，诱导细胞的凋亡，并阻滞细胞周期；CK18还可能通过诱导EMT发生参与乳腺癌 BT549细胞的侵袭转移过程。

关键词: 角蛋白18, 乳腺肿瘤, 细胞增殖, 细胞凋亡

Abstract: ponding Author E-mail: gl-gump Abstract: Objective To investigate the effects of cytokeratin 18 (CK18) gene on proliferation, apoptosis and invasion of human breast cancer BT549 cells, and to explore the molecular mechanism. Methods CK18 stably silencing BT549 cell line was established and divided into blank control group, negative control group and CK18-shRNA group. The cells with CK18 target silencing were used as the CK18-shRNA group, the empty vector was used as the negative control group (sh Con), and the blank control group (Wt) was not treated. The established cell line was identified by Western blot assay (WB). Cell proliferation was detected by cell counting kit-8 (CCK-8) assay and colony formation assay. Cell apoptosis was analyzed by flow cytometry of Annexin V/PE double staining kit, and cell cycle analysis was detected by PI-FACS. Migration and invasion capability were measured by the Scratch assay and Transwell assay. Western blot assay was used to detect the metastasis-related molecules of E-cadherin and vimentin protein expression. Results CK18 silencing BT549 cell line was established. The WB result indicated that the expression of CK18 was significantly inhibited, and the inhibition rate was up to 73%. It was shown that the BT549 cell proliferation was obviously depressed in the time-dependent manner at the time points of 24 h, 48 h and 72 h in CK18-shRNA group with the comparison to blank control group and negative control group. Compared with the blank control group and negative control group, the migration and invasion ability were decreased in CK18-shRNA group, and the apoptosis rate and the ratio of cells in G2 phase were increased in CK18-shRNA group (P＜ 0.05). WB showed that the decreased expression of CK18 also promoted the expression of E-cadherin and depressed the expression of vimentin (P＜0.05). Conclusion CK18 gene silencing in BT549 cells can inhibit cell proliferation, migration and invasion, and induce apoptosis and arrest cell cycles. CK18 may play a role in metastasis process of breast cancer BT549 cells by inducing EMT.

Key words: keratin 18;, breast neoplasms, cell proliferation, apoptosis