应用CRISPR/Cas 9系统构建肺癌细胞系获得AMPKα1基因敲除的稳定细胞株

Application of CRISPR/Cas 9 System to Construct Lung Cancer Cell Lines to Obtain Stable Cell Lines with AMPKα1 Knockdown

目的利用CRISPR/Cas 9系统在人肺癌细胞系A549和H460中获得敲除腺苷单磷酸活化蛋白激酶α1(adenosine monphosphate-activated protein kinase,AMPKα1)的稳定表达细胞系。方法根据CRISPR/Cas 9靶点设计原则,设计三条引导RNA(sgRNA),分别构建AMPKα1-CRISPR/Cas9 KO及其HDR质粒。将质粒转染至A549和H460细胞后,筛选稳定的单克隆细胞株。用Western blot鉴定筛选获得的肺癌细胞株是否表达AMPKα1。实验被分为原始对照组和敲除组,采用MTT法检测AMPKα1的肺癌细胞增殖情况。结果经过CRISPR/Cas 9系统处理后,筛选获得的A549和H460敲除AMPKα1稳定细胞株与原始细胞株A549和H460相比,Western blot检测不到AMPKα1蛋白质的表达,差异具有统计学意义(t=137.7,79.17,均P<0.0001)。MTT结果显示,筛选获得的A549和H460敲除AMPKα1的稳定细胞株与原始细胞株A549和H460相比,敲除细胞株的增殖速度明显减弱,差异具有统计学意义(t=3.956~28.16,均P<0.05)。结论CRISPR/Cas 9系统成功建立敲除AMPKα1表达的稳定肺癌细胞株,为进一步研究AMPKα1在肺癌发生发展中的作用提供细胞模型。

Objective To construct stably lung cancer cell lines that did not express adenosine monphosphate-activated protein kinaseα1(AMPKα1)using CRISPR/Cas 9 system.Methods According to the CRISPR/Cas 9 target design principle,three small guide RNA(sgRNAs)were designed to construct AMPKα1-CRISPR/Cas 9 KO and its HDR plasmid,respectively.After the plasmids were transfected into A549 and H460 cells,stable monoclonal cell lines were selected.The expression of AMPKα1was detected in stable monoclonal cell lines by Western blot.The experiments were divided into original control and knockout groups,MTT assay was used to detect the proliferation of AMPKα1 knockout lung cancer cells.Results After treatment with the CRISPR/Cas 9 system,the knockdown AMPKα1 stable cell lines obtained from the screening were undetectable by Western blot for AMPKα1 protein expression compared with the original cell lines A549 and H460,and the differences were statistically significant(t=137.7,79.17,all P<0.0001).The MTT results showed that the proliferation rate of knockdown cell lines obtained from the screened stable cell lines of A549 and H460 knockdown AMPKα1 was significantly weaker compared with the original cell lines A549 and H460,and the differences were statistically significant(t=3.956~28.16,all P<0.05).Conclusion The stable lung cancer cell lines with no expression of AMPKα1 were established by using CRISPR/CAS 9system.It provides a cellular model for further studies on the role of AMPKα1 in lung carcinogenesis and development.