TSA上调miR-4298靶向抑制PADI4表达在诱导U251细胞凋亡中的作用  

Roles of TSA upregulation miR-4298 targeting inhibition of PADI4 expression in inducing U251 cells apoptosis

作　　者：王宪伟 史美燕 王凤芹 齐福 王朝喆 周飞 Wang Xianwei;Shi Meiyan;Wang Fengqin;Qi Fu;Wang Chaozhe;Zhou Fei(Department of Neurology,Jining Wenshang County People′s Hospital of Shandong Province,Jining 272501,China;Institute of Immunology and Biotechnology Transformation,Binzhou Medical College,Yantai 256600,China;Institute of Basic Medicine,Shandong Academy of Medical Sciences&Shandong First Medical University,Jinan 250062,China)

机构地区：[1]山东省济宁市汶上县人民医院神经内科,济宁272501 [2]滨州医学院免疫与生物技术转化研究院,烟台256600 [3]山东第一医科大学(山东省医学科学院)基础医学研究所,济南250062

出　　处：《国际肿瘤学杂志》2021年第4期193-199,共7页Journal of International Oncology

基　　金：山东省自主成果转化重大专项(2015ZDJS04003)。

摘　　要：目的探讨微小RNA-4298(miR-4298)靶向抑制肽酰精氨酸脱亚胺基酶4(PADI4)表达在组蛋白去乙酰化酶抑制因子曲古抑菌素A(TSA)诱导U251细胞凋亡中的作用机制。方法实验分为TSA组(0.2μmol/L TSA处理)和对照组。采用CCK8法检测TSA对U251细胞增殖的影响;流式细胞术检测药物作用后U251细胞的凋亡水平;反转录PCR和蛋白质印迹法测定miR-4298干扰后PADI4基因和蛋白表达的变化;Luciferase实验测定miR-4298对PADI4的3′UTR区靶向结合与荧光素酶活性的影响;PADI4转染U251细胞,观察miR-4298对凋亡功能的拯救作用。实验各分为3组,即NC组、miR-4298 mimic组、TSA+miR-4298 mimic组以及NC组、miR-4298 inhibitor组、TSA+miR-4298 inhibitor组。结果U251细胞经0.2μmol/L的TSA作用4 d后,对照组的吸光度(A)值为1.168±0.148,高于TSA组的0.737±0.007,差异有统计学意义(t=4.948,P=0.008)。与对照组相比,经0.2μmol/L TSA处理48 h后,U251细胞发生明显的凋亡现象(27.62%±3.49%vs.4.99%±0.13%,t=11.190,P<0.001)。与药物作用前相比,TSA作用48 h后,PADI4基因表达(0.386±0.020 vs.0.903±0.021)和蛋白表达水平(0.276±0.041 vs.0.777±0.031)均有所降低,差异均有统计学意义(t=30.400,P<0.001;t=16.770,)P<0.001。miR-4298在TSA诱导U251细胞凋亡的过程中表达升高(2.573±0.289 vs.1.003±0.136;t=8.487,P=0.001),并且miR-4298与PADI4的表达呈负相关(r=-0.877,P=0.002)。Luciferase实验证实,miR-4298对野生型PADI4的3′UTR区具有靶向结合和荧光素酶抑制作用。与NC组(0.920±0.026)相比,miR-4298 mimic组(0.413±0.049)和TSA+miR-4298 mimic组(0.213±0.035)PADI4基因相对表达水平均有所降低,差异均有统计学意义(均P<0.001);其蛋白表达水平变化与基因变化趋势一致。与NC组(3.78%±0.68%)相比,miR-4298 mimic组(7.96%±1.10%)和TSA+miR-4298 mimic组(13.74%±1.26%)诱导细胞凋亡的水平均有所增加,差异有统计学意义(P=0.005;P<0.001)。与NC组(0.183±0.025)相比,miR-4298 inhibitor组(0.483±0Objective To investigate the mechanism of microRNA-4298(miR-4298)targeting inhibition of peptidylarginine deiminase 4(PADI4)expression in U251 cells apoptosis induced by histone deacetylase inhibitor trichostatin A(TSA).Methods The experiment was divided into TSA group(0.2μmol/L TSA)and contral group.CCK8 method was used to detect the effect of TSA on the proliferation of U251 cells.Flow cytometry was used to detect the apoptosis level of U251 cells after drug action.Reverse transcription PCR and Western blotting experiments were used to determine the changes of PADI4 gene and protein expression after miR-4298 interference.Luciferase assay was used to determine the effects of miR-4298 on targeted binding and luciferase activity in the 3′UTR region of PADI4.U251 cells were transfected with PADI4 to observe the rescue effect of miR-4298 on apoptosis.Each experiment was divided into 3 groups,NC group,miR-4298 mimic group,TSA+miR-4298 mimic group and NC group,miR-4298 inhibitor group,TSA+miR-4298 inhibitor group.Results U251 cells were treated with 0.2μmol/L TSA for 4 days,the Absorbancy(A)values of the control group was 1.168±0.148,which was higher than those of the TSA group(0.737±0.007),with statistically significant difference(t=4.948,P=0.008).Compared with the control group,after treatment with 0.2μmol/L TSA for 48 h,U251 cells showed obvious apoptosis(27.62%±3.49%vs.4.99%±0.13%,t=11.190,P<0.001).Compared with those before the drug treatment,the PADI4 gene expression(0.386±0.020 vs.0.903±0.021)and protein expression(0.276±0.041 vs.0.777±0.031)after TSA treatment for 48 h both were decreased,with statistically significant differences(t=30.400,P<0.001;t=16.770,P<0.001).The expression of miR-4298 during the process of TSA-induced U251 cell apoptosis increased(2.573±0.289 vs.1.003±0.136;t=8.487,P=0.001).There was a negative correlation between the miR-4298 expression and PADI4 expression(r=-0.877,P=0.002).Luciferase experiment confirmed that miR-4298 has targeted binding and luciferase inhibitory effe

关 键 词：神经胶质瘤 细胞凋亡 曲古抑菌素A 肽酰基精氨酸脱亚胺酶4 miR-4298 

分 类 号：R739.41[医药卫生—肿瘤]