慢病毒介导的TET2基因稳定敲低SKM-1细胞株的构建及验证

Construction and Validation of Lentivirus⁃mediated Stable Knockdown of SKM⁃1 in TET2 Gene

目的:构建稳定敲低TET2基因的SKM-1细胞株,将包装好的H_TET2-shRNA慢病毒感染SKM-1细胞株,得到H_TET2-shRNA SKM-1稳定株。方法:将稳定敲低的SKM-1细胞株进行qPCR和Western blot实验验证。利用CCK-8细胞活力检测试剂盒和流式细胞术分别检测转染前后SKM-1细胞的增殖活性和凋亡情况。结果:qPCR验证H_TET2-shRNA的SKM-1细胞中TET2基因表达量明显降低;Western blot表明H_TET2-shRNA的SKM-1细胞中TET2蛋白表达量也明显降低;利用CCK-8细胞活力检测证实了TET2基因稳定敲低后并不影响细胞的活力;细胞周期检测发现敲低TET2后S期细胞占总细胞数的48.1%,高于SKM-1组(42.3%)和对照空质粒组;细胞凋亡检测发现TET2敲低组SKM-1细胞凋亡率为0.6%,低于空质粒组的4.4%。结论:通过慢病毒介导构建TET2基因稳定低表达的SKM-1细胞株,利用PCR、Western blot实验验证成功构建细胞株,利用CCK-8细胞活力检测证实TET2基因稳定敲低后并不影响细胞活力;流式细胞术细胞周期检测发现TET2基因敲低会影响细胞DNA复制功能,导致细胞间期分裂异常,影响细胞生长和自我修复;流式细胞术细胞凋亡检测发现TET2基因敲低会抑制细胞凋亡,这可能与TET2是抑癌基因相关。

Objective:To construct the SKM⁃1 cell line with stable knockdown of the TET2 gene,and to infect SKM⁃1 cell line with the packaged H_TET2⁃shRNA lentivirus to obtain the H_TET2⁃shRNA SKM⁃1 stable strain.Methods:The SKM⁃1 cell line with stable knockdown of TET2 gene was verified by qPCR and Western blot(WB)experiments.The proliferative activity and apoptosis of SKM⁃1 cells before and after transfection were detected using the cell counting kit⁃8(CCK⁃8)cell viability assay and flow cytometry,respectively.Results:qPCR verified that the TET2 gene expression was significantly reduced in SKM⁃1 cells with H_TET2⁃shRNA.Western blot showed that the TET2 protein expression was also significantly reduced in SKM⁃1 cells with H_TET2⁃shRNA and the CCK⁃8 cell viability assay confirmed that stable knockdown of the TET2 gene did not affect cell viability.The cell cycle assay revealed that S⁃phase cells accounted for 48.1%of the total cell number after knockdown of TET2,which was higher than that of the SKM⁃1 group(42.3%)and the control empty plasmid group.The apoptosis assay revealed that the apoptosis rate of SKM⁃1 cells in the TET2 knockdown group was 0.6%lower than that of the empty plasmid group(4.4%).Conclusions:SKM⁃1 cell line with stable low expression of the TET2 gene was constructed by lentivirus⁃mediated transfection,and the constructed cell line was verified to be successful by using qPCR and WB experiments.It was confirmed that the stable knockdown of TET2 did not affect the viability of the cells by using the CCK⁃8 cell viability assay.The cell cycle assay of flow cytometry found that the knockdown of TET2 affected the function of cellular DNA replication,which led to the abnormalities of cellular interphase division and affected cell growth and self⁃repair.The flow cytometry apoptosis assay found that knockdown of TET2 would inhibit apoptosis,which may be related to the fact that TET2 is an anti⁃oncogene.