生物学杂志

生物学杂志 ›› 2019, Vol. 36 ›› Issue (6): 25-.doi: 10.3969/j.issn.2095-1736.2019.06.025

• 研究报告 • 上一篇    下一篇

敲除PLAC8蛋白对人胚肾细胞增殖的影响

  

  1. 中南民族大学 生命科学学院 医学生物研究所 武陵山区特色资源植物种质保护与利用湖北省重点实验室， 武汉 430074
  • 出版日期:2019-12-18 发布日期:2019-12-12
  • 通讯作者: 薛璐，博士研究生，副教授，研究方向为分泌蛋白在肿瘤发生发展中的分子功能机制，E-mail：3097910@scuec.edu.cn
  • 作者简介:秦绪慧，硕士研究生，专业方向为分泌蛋白在肿瘤发生发展中的分子功能机制，E-mail：1045180249@qq.com
  • 基金资助:
    湖北省自然科学基金一般面上项目（No. 2018CFB594）

The effects of PLAC8 knockout on proliferation of human embryonic kidney cell line 293T

  1. Institute for Medical Biology and Hubei Provincial Key Laboratory for Protection and Application of Special Plants in Wuling Area of China, College of Life Sciences, South-Central University for Nationalities, Wuhan 430074, China
  • Online:2019-12-18 Published:2019-12-12

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摘要：

为了构建PLAC8蛋白敲除的人胚肾细胞株 (293T)并检测其对293T细胞增殖的影响，基于CRISPR/Cas9基因编辑技术及流式细胞分选术构建敲除PLAC8蛋白的293T细胞株，通过基因组测序及错配酶酶切进行编辑细胞系的筛选。用筛选得到的细胞系提取细胞总蛋白，通过蛋白免疫印迹法 (Western Blot)检测编辑效率。进一步通过细胞生存实验 (MTT实验)检测敲除PLAC8对293T细胞增殖的影响，通过蛋白免疫印迹法 (Western Blot)检测敲除PLAC8蛋白后293T细胞中增殖相关基因的表达水平变化。结果表明，成功构建了PLAC8 蛋白敲除的细胞系。与野生型细胞系相比，PLAC8表达下调会抑制细胞增殖，且在这一过程中AKT、RAF-1、ERK2、C-MYC等4个与增殖相关基因的蛋白水平改变，提示PLAC8可能通过AKT及RAF-1-ERK2-C-MYC这一级联通路信号调控细胞增殖。


关键词: CRISPR/Cas9, 基因敲除, PLAC8, 细胞增殖, MAPK信号通路

Abstract:

In order to construct Placenta special gene 8 (PLAC8) knockout human embryonic kidney cell line (293T) and detect the effect of PLAC8 knockout on cell proliferation, based on CRISPR/Cas9 and flow cytometry technology, a PLAC8 knockout 293T cell line was constructed. Edited cells were screened by genome sequencing and T7E1 enzyme digestion. The total protein of the selected cells was extracted to measure the efficiency of editing by Western Blot. Furthermore, cell survival experiment (MTT assay) was employed to measure the effect of PLAC8 knockout on cell proliferation. Meanwhile, the expression levels of the proliferation-related genes in PLAC8 knockout 293T cells were detected via Western Blot. In conclusion， we successfully constructed PLAC8-knockout cell line. Compared with wildtype 293T, knockout of PLAC8 could inhibit cell proliferation. The decreased cell proliferation was associated with the reduced expression of AKT and C-MYC and increased expression of RAF-1 and ERK2, which indicated that PLAC8 might regulate cell proliferation via AKT and RAF-1-ERK2-C-MYC cascade signal pathway


Key words: CRISPR/Cas9, knockout, PLAC8, cell proliferation, MAPK signal pathway

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