摘要
目的探讨DNA损伤诱导转录因子4(DDIT4)在肺腺癌细胞中的表达及其对细胞增殖、凋亡的影响和可能机制。方法实时荧光定量PCR(qRT-PCR)和蛋白质印迹检测DDIT4在肺腺癌细胞系(H1299和A549)及正常肺支气管上皮细胞(BEAS-2B)中表达;慢病毒转染抑制A549细胞中DDIT4表达,分为DDIT4敲低慢病毒(sh-DDIT4,分为sh-DDIT4#1、sh-DDIT4#2和sh-DDIT4#3)组和转染空病毒(NC)组。蛋白质印迹检测细胞中DDIT4沉默效果;CCK-8、平板克隆形成实验检测sh-DDIT4对肺腺癌细胞增殖能力的影响;TUNEL凋亡实验检测sh-DDIT4对肺腺癌细胞凋亡能力的影响;Transwell和划痕实验检测sh-DDIT4对肺腺癌细胞侵袭和迁移能力的影响。结果qRT-PCR结果显示,DDIT4 mRNA在BEAS-2B、H1299和A549细胞中相对表达水平分别为1.01±0.04、2.19±0.09和3.10±0.09,差异有统计学意义,F=183.00,P<0.001。蛋白质印迹结果显示,DDIT4蛋白在BEAS-2B、H1299和A549中表达水平分别为0.39±0.02、0.98±0.01和1.00±0.02,差异有统计学意义,F=457.00,P<0.001。DDIT4mRNA在NC、sh-DDIT4#1、sh-DDIT4#2和sh-DDIT4#3组中表达量分别为1.01±0.02、0.58±0.02、0.59±0.02和0.27±0.04,差异有统计学意义,F=150.40,P<0.001;DDIT4蛋白在各组中表达量分别为1.12±0.01、0.61±0.02、0.62±0.02和0.23±0.05,差异有统计学意义,F=145.10,P<0.001。CCK-8增殖实验结果显示,120h时NC和sh-DDIT4组细胞生长率分别为0.91±0.04和0.57±0.01,差异有统计学意义,t=7.71,P<0.001。克隆形成实验结果显示,NC和sh-DDIT4组细胞克隆形成数量分别为120.70±6.64和10.67±0.88,差异有统计学意义,t=16.42,P<0.001。EdU实验结果显示,NC和sh-DDIT4组细胞的EdU阳性率分别为(64.33±1.76)%和(13.33±1.20)%,差异有统计学意义,t=23.89,P<0.001。细胞凋亡结果显示,NC和sh-DDIT4组细胞凋亡率分别为(22.33±1.45)%和(49.67±0.88)%,差异有统计学意义,t=16.08,P<0.001。细胞划痕实验结果显示,36h时NC和sh-DDIT4组细胞愈合度分别(84.33±1.86)%和(44.67±2.03)%,差异有统计学意义,t=14.43,P<0.001。Transwell侵袭实验结果显示,NC和sh-DDIT4组穿过小室的细胞数量分别为为(118.00±3.79)和(28.00±1.73)个,差异有统计学意义,t=21.62,P<0.001。结论DDIT4在肺腺癌细胞中表达上调,沉默DDIT4表达可抑制肺腺癌细胞增殖、迁移及侵袭,诱导细胞凋亡。
Objective To investigate the expression of DNA damage-induced transcription factor 4(DDIT4)in lung adenocarcinoma(LUAD)and its effect on cell proliferation and apoptosis and its possible mechanism.Methods The expression of DDIT4in LUAD cell lines(H1299and A549)and normal lung bronchial epithelial cells(BEAS-2B)was detected by real-time fluorescence quantitative PCR(qPCR)and Western blotting.The expression of DDIT4in A549cells was inhibited by lentivirus transfection.They were divided into DDIT4knockdown lentivirus(Sh-DDIT4)group and transfection empty virus(NC)group.Western blotting was used to detect DDIT4silencing effect in cells.The effects of Sh-DDIT4on the proliferation of LUAD cells were detected by CCK-8and plate clonal formation assay.TUNEL apoptosis assay was used to detect the effect of Sh-DDIT4on apoptosis of LUAD cells.Transwell and scratch assay were used to detect the effects of Sh-DDIT4on the invasion and migration of LUAD cells.Results qRT-PCT results showed that the relative expression levels of DDIT4mRNA in BEAS-2B,H1299and A549cells were 1.01±0.04,2.19±0.09and 3.10±0.09,respectively,and the difference was statistically significant(F=183.00,P<0.001).Western blotting results showed that DDIT4protein expression levels in BEAS-2B,H1299and A549were 0.39±0.02,0.98±0.01and 1.00±0.02,and the difference was statistically significant(F=457.00,P<0.001).A549cells were transfected into NC group,Sh-DDIT4#1,Sh-DDIT4#2and Sh-DDIT4#3groups,respectively.The expression levels of DDIT4mRNA in NC group,Sh-DDIT4#1,Sh-DDIT4#2and Sh-DDIT4#3groups were 1.01±0.02,0.58±0.02,0.59±0.02and 0.27±0.04,respectively,and the difference was statistically significant(F=150.40,P<0.001).The expression levels of DDIT4protein in NC group and Sh-DDIT4#1,Sh-DDIT4#2and Sh-DDIT4#3groups were 1.12±0.01,0.61±0.02,0.62±0.02and 0.23±0.05,respectively,and the differences were statistically significant(F=145.10,P<0.001).CCK-8proliferation test results showed that the cell growth rates of NC group and Sh-DDIT 4group at 120hwere 0.91±0.04and 0.57±0.01,respectively,and the difference was statistically significant(t=7.71,P<0.001).The results of clone formation experiment showed that the number of cell clone formation in NC group and Sh-DDIT 4group was 120.70±6.64and 10.67±0.88,respectively,and the difference was statistically significant(t=16.42,P<0.001).EdU test results showed that the positive rates of EdU in NC group and Sh-DDIT 4group were(64.33±1.76)%and(13.33±1.20)%,respectively,and the difference was statistically significant(t=23.89,P<0.001).The apoptosis rates of NC group and Sh-DDIT 4group were(22.33±1.45)%and(49.67±0.88)%,respectively,the difference was statistically significant(t=16.08,P<0.001).The results of cell scratch test showed that the healing degree of NC group and Sh-DDIT 4group at 36hwas(84.33±1.86)%and(44.67±2.03)%,respectively,and the difference was statistically significant(t=14.43,P<0.001).Results of Transwell invasion assay showed that the number of cells passing through the chamber in NC group and Sh-DDIT 4 group was 118.00±3.79and 28.00±1.73,respectively,and the difference was statistically significant(t=21.62,P<0.001).Conclusion The expression of DDIT4was up-regulated in LUAD cells.Silencing DDIT4expression can inhibit the proliferation,migration and invasion of LUAD cells,and induce cell apoptosis.
作者
栾艳超
彭海军
韩青松
白峰
王明正
LUAN Yan-chao;PENG Hai-jun;HAN Qing-song;BAI Feng;WANG Ming-zheng(Hebei Key Laboratory of Lung Disease,Hebei Chest Hospital,Shijiazhuang050001,China)
出处
《中华肿瘤防治杂志》
CAS
北大核心
2022年第20期1460-1466,共7页
Chinese Journal of Cancer Prevention and Treatment
基金
河北省卫生健康委科研基金项目(20191004)
2019年度河北省财政厅省直医疗卫生机构老年病防治项目。
关键词
肺肿瘤
腺癌
细胞增殖
细胞凋亡
细胞运动
DDIT4
lung neoplasms
adenocarcinoma
cell proliferation
apoptosis
cell movement
DNA damage-induced transcription factor 4
