建立稳定敲减MINK1表达的肺腺癌细胞系及对其初步分析

Establishment and preliminary analysis of lung adenocarcinoma cell lines with stable MINK1 knockdown

目的:构建稳定敲减Misshapen/NIK-related kinase(MINK1)表达肺腺癌细胞系,并检测稳定敲减其表达后对细胞增殖、迁移运动及顺铂治疗的影响。方法:利用肺腺癌A549和PC-9细胞,基于pLKO.1-shRNA慢病毒基因敲减表达系统构建稳定敲减MINK1表达细胞系,分别通过qRT-PCR和Western bolt在mRNA和蛋白水平检测敲减效率。CCK-8和Transwell检测稳定敲减MINK1表达对细胞增殖和迁移运动的影响,流式细胞检测细胞周期与凋亡变化情况;分析稳定敲减MINK1表达对顺铂治疗敏感性变化情况,并通过免疫荧光染色DNA损伤标志物γH2AX检测细胞DNA损伤水平。结果:qRT-PCR和Western bolt分析结果显示2个特异shRNA均可显著下调MINK1表达。稳定敲减MINK1表达的肺腺癌细胞增殖能力降低,迁移运动能力下降;且敲减MINK1表达明显增强化疗药物顺铂对癌细胞杀伤作用,并诱导DNA损伤水平增加。结论:利用肺腺癌A549和PC-9细胞成功构建稳定敲减MINK1肺腺癌细胞系,而稳定敲减其表达可抑制癌细胞增殖及迁移运动,且可能通过调节DNA损伤修复过程促肺癌细胞耐药。

Objective:To establish lung adenocarcinoma cell lines with stable knockdown of the expression of encoding Misshapen/NIK-related kinase(MINK1)and analyze the effects of stable MINK1 knockdown on cell proliferation,migration,and cisplatin treat⁃ment.Methods:In lung adenocarcinoma A549 and PC-9 cells,cell lines with stable MINK1 knockdown were constructed based on the pLKO.1-shRNA lentiviral expression system.The knockdown efficiency was detected at the mRNA and protein levels using qRT-PCR and Western blot.CCK-8 and Transwell assays were used to assess the effects of stable MINK1 knockdown on cell proliferation and mi⁃gration.Flow cytometry was used to detect changes in cell cycle and apoptosis.The sensitivity of cells with stable MINK1 knockdown to cisplatin treatment was analyzed.The level of DNA damage in cells was detected by immunofluorescence staining of the DNA damage markerγH2AX.Results:qRT-PCR and Western blot showed that shRNA significantly downregulated the expression of MINK1.Prolif⁃eration and migration were reduced in lung adenocarcinoma cells with stable MINK1 knockdown.MINK1 knockdown significantly en⁃hanced the cytotoxic effect of the chemotherapy drug cisplatin on cancer cells and induced an increase in the level of DNA damage.Conclusion:Lung adenocarcinoma cell lines with stable MINK1 knockdown were successfully established using A549 and PC-9 cells.Stable MINK1 knockdown can inhibit cancer cell proliferation and migration and may reduce the drug resistance of lung cancer cells by regulating the DNA damage repair process.