MLCK敲除对FaDu细胞凋亡的影响

Effect of MLCK knockout on apoptosis in FaDu cells

目的构建肌球蛋白轻链激酶(MLCK)敲除的下咽癌FaDu细胞株,并探讨敲除MLCK对FaDu细胞凋亡的影响。方法脂质体转染sgRNA和Cas92NLS Nuclease构建MLCK敲除的FaDu细胞株,提DNA测序确定敲除细胞株,分为对照组、MLCK KO 1组、MLCK KO 2组;采用RT-qPCR、Western blot检测MLCK敲除效率;流式细胞术检测MLCK敲除对细胞周期和凋亡的影响;Western blot检测MLCK敲除对细胞凋亡的影响。结果DNA测序显示在sgRNA序列识别处,MLCK碱基序列发生了缺失或替换;RT-qPCR和Western blot显示MLCK敲除细胞株mRNA和蛋白质水平低于对照组(P<0.0001);流式细胞术实验显示敲除MLCK对FaDu细胞的周期无明显变化,但凋亡率增加(P<0.0001);Western blot检测显示,MLCK敲除组Bax/Bcl-2(P<0.0001)和Cleaved Caspase-3/Caspase-3(P=0.0007)增加。结论敲除MLCK诱导细胞凋亡,但具体机制需进一步研究。

Objective To construct a myosin light chain kinase(MLCK)knockout hypopharyngeal carcinoma FaDu cell line and discuss the knockout effect of MLCK on apoptosis of FaDu cells.Methods MLCK knockout FaDu cell lines were constructed by liposome transfection of sgRNA and Cas92NLS Nuclease,and the knockout cell lines were identified by DNA sequencing and divided into control group,MLCK KO 1 group and MLCK KO 2 group.The knockout efficiency of MLCK was assessed by RT-qPCR and Western blot,and the effect of MLCK knockout on cell cycle and apoptosis was detected by flow cytometry while Western bolt was conducted to detect the effect of MLCK knockout on cell apoptosis.Results DNA sequencing showed that MLCK base sequences were missing or substituted at the sgRNA sequence recognition,and RT-qPCR and Western blot showed lower mRNA and protein levels in MLCK knockout cell lines than those in the control group(P<0.0001).Furthermore,the flow cytometry assay showed that MLCK knockout did not significantly change the cycle of FaDu cells,but the rate of apoptosis increased(P<0.0001).Western blot showed that Bax/Bcl-2(P<0.0001)and Cleaved Caspase-3/Caspase-3(P=0.0007)ratios raised in the MLCK knockout group.Conclusion The knockout of MLCK induced apoptosis in FaDu cells,but the exact mechanism needs further study.