利用CRISPR/Cas9技术建立PTEN敲除的人子宫内膜腺癌细胞模型及其功能研究

CRISPR/Cas9-based PTEN knockout in human endometrial adenocarcinoma cells

目的利用CRISPR/Cas9技术构建靶向PTEN的基因编辑质粒,构建PTEN稳定敲除的人子宫内膜腺癌细胞株KLE,并初步探讨PTEN敲除对子宫内膜癌细胞生物学功能的影响。方法设计靶向人PTEN基因的sgRNA序列,构建puro-cas9-PTENsgRNA质粒,测序验证;将重组质粒转染人子宫内膜腺癌细胞KLE,嘌呤霉素筛选获得PTEN稳定敲除细胞株;用T7E1酶切实验检测切割效率,Western blotting检测PTEN的蛋白表达水平,并检测细胞自噬相关因子LC3、P62的表达水平,检测细胞活性,探讨PTEN敲除对子宫内膜癌细胞自噬的调控作用。结果 CRISPR/Cas9系统对KLE细胞基因组中的PTEN进行了切割,并在同源重组时发生了碱基错配,PTEN被成功敲除;PTEN敲除降低了细胞自噬相关因子LC3Ⅰ型向Ⅱ型转化,增加了P62的累积。PTEN敲除的细胞活性增强。结论利用CRISPR/Cas9基因编辑技术,在人子宫内膜腺癌细胞中成功敲除PTEN,抑制了人子宫内膜腺癌细胞的自噬水平,为后续研究提供了细胞模型。

Objective Using the CRISPR/Cas9 system,we constructed a gene editing plasmid targeting PTEN and stably knocked out PTEN in the human endometrial adenocarcinoma cell line KLE.The regulation of the PTEN knockout on the biological function in endometrial cancer cells was investigated.Methods A sgRNA sequence targeting the human PTEN gene was designed and the purocas9-PTEN-sgRNA plasmid was constructed.KLE human endometrial adenocarcinoma cells were transfected with the recombinant plasmid.Using purinomycin screening,we obtained a PTEN-stable knockout cell line.The T7 endonuclease 1 was used to detect cutting efficiency.Western blotting was used to detect PTEN protein levels and the expression of autophagy related factors LC3 and P62,as well as cell activity,to explore the regulation of PTEN on autophagy in endometrial cancer cells.Results We successfully adapted the CRISPR/Cas9 system to edit the KLE genome and knockout PTEN due to base mismatch during homologous recombination.PTEN knockout decreased in conversion of LC3-Ⅰto LC3-Ⅱand increased the accumulation of P62.PTEN knockout cells showed increased cell activity.Conclusion Using the CRISPR/Cas9 gene editing technique,PTEN could be successfully knocked out in human endometrial adenocarcinoma cells and inhibit autophagy levels in these cells.We provide a cell model for subsequent studies.