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

doi:10.3760/cma.j.cn371439-20201011-00039

国际肿瘤学杂志 ›› 2021, Vol. 48 ›› Issue (4): 193-199.doi: 10.3760/cma.j.cn371439-20201011-00039

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

王宪伟¹, 史美燕¹(), 王凤芹¹, 齐福², 王朝喆², 周飞³

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

收稿日期:2020-10-11 修回日期:2021-02-01 出版日期:2021-04-08 发布日期:2021-06-18
通讯作者: 史美燕 E-mail:shimeiyan715@163.com
基金资助:
山东省自主成果转化重大专项(2015ZDJS04003)

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

Wang Xianwei¹, Shi Meiyan¹(), Wang Fengqin¹, Qi Fu², Wang Chaozhe², Zhou Fei³

¹Department of Neurology, Jining Wenshang County People's Hospital of Shandong Province, Jining 272501, China
²Institute of Immunology and Biotechnology Transformation, Binzhou Medical College, Yantai 256600, China
³Institute of Basic Medicine, Shandong Academy of Medical Sciences & Shandong First Medical University, Jinan 250062, China

Received:2020-10-11 Revised:2021-02-01 Online:2021-04-08 Published:2021-06-18
Contact: Shi Meiyan E-mail:shimeiyan715@163.com
Supported by:
Major Special Project of Independent Achievements Transformation of Shandong Province(2015ZDJS04003)

摘要/Abstract

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(0.2 μmol/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0.2 μmol/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0.2 μmol/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±0.020vs. 0.903±0.021)和蛋白表达水平(0.276±0.041vs. 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±0.289vs. 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±0.032)和TSA+miR-4298 inhibitor组(0.386±0.025)PADI4基因表达均有所升高,差异有统计学意义(P<0.001;P=0.015)。蛋白表达水平变化与基因变化趋势一致。与NC组(4.96%±0.59%)相比,miR-4298 inhibitor组(23.83%±2.20%)和TSA+miR-4298 inhibitor组(9.55%±1.49%)诱导细胞凋亡的水平均有所增加,差异有统计学意义(均P<0.001)。救援实验中,miR-4298 mimic组明显升高PADI4表达水平(P<0.001),miR-4298 mimic+PADI4组则相对逆转PADI4表达水平(P=0.002)。结论 miR-4298可通过靶向调控PADI4基因表达参与U251细胞的凋亡发生过程,miR-4298可能是脑胶质瘤靶向干预治疗的靶点。

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

Abstract:

Objective 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 (0.2 μmol/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 0.2 μmol/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 0.2 μmol/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±0.020vs. 0.903±0.021) and protein expression (0.276±0.041vs. 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±0.289vs. 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 effects on the 3'UTR region of wild-type PADI4. Compared with NC group (0.920±0.026), the relative expression levels of PADI4 gene of miR-4298 mimic group (0.413±0.049) and TSA+miR-4298 mimic group (0.213±0.035) were decreased, with statistically significant differences (allP<0.001). The PADI4 protein expression level change was consistent with the gene change trend. Compared with NC group (3.78%±0.68%), the apoptosis levels of miR-4298 mimic group (7.96%±1.10%) and TSA+miR-4298 mimic group (13.74%±1.26%) were increased, with statistically significant differences (P=0.005; P<0.001). Compared with NC group (0.183±0.025), the PADI4 gene expression of miR-4298 inhibitor group (0.483±0.032) and TSA+miR-4298 inhibitor group (0.386±0.025) were increased, with statistically significant differences (P<0.001; P=0.015). The PADI4 protein expression level change was consistent with the gene change trend. Compared with NC group (4.96%±0.59%), the apoptosis levels of miR-4298 inhibitor group (23.83%±2.20%) and TSA+miR-4298 inhibitor group (9.55%±1.49%) were increased, with statistically significant differences (allP<0.001). In the rescue experiment, the expression level of PADI4 in miR-4298 mimic group was significantly increased (P<0.001), while the expression level of PADI4 in miR-4298 mimic+PADI4 group was relatively reversed (P=0.002). Conclusion miR-4298 can participate in the process of U251 cell apoptosis by targeting the expression of PADI4 gene, and miR-4298 may be the target of targeted intervention therapy for glioma.

Key words: Glioma, Apoptosis, Trichostatin A, Peptidylarginine deiminase 4, miR-4298

王宪伟, 史美燕, 王凤芹, 齐福, 王朝喆, 周飞. TSA上调miR-4298靶向抑制PADI4表达在诱导U251细胞凋亡中的作用[J]. 国际肿瘤学杂志, 2021, 48(4): 193-199.

Wang Xianwei, Shi Meiyan, Wang Fengqin, Qi Fu, Wang Chaozhe, Zhou Fei. Roles of TSA upregulation miR-4298 targeting inhibition of PADI4 expression in inducing U251 cells apoptosis[J]. Journal of International Oncology, 2021, 48(4): 193-199.

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组别	0 d	1 d	2 d	3 d	4 d
对照组	0.378±0.030	0.556±0.020	0.754±0.057	0.889±0.051	1.168±0.148
TSA组	0.380±0.021	0.459±0.026	0.536±0.034	0.617±0.075	0.737±0.007
t值	0.602	5.003	5.700	5.143	4.948
P值	0.580	0.008	0.005	0.007	0.008

组别	PADI4
组别	基因	蛋白
NC组	0.920±0.026	0.846±0.070
miR-4298 mimic组	0.413±0.049^a	0.533±0.041^b
TSA+miR-4298 mimic组	0.213±0.035^a	0.216±0.040^a
F值	273.466	103.841
P值	<0.001	<0.001

组别	PADI4
组别	基因	蛋白
NC组	0.183±0.025	0.146±0.021
miR-4298 inhibitor组	0.483±0.032^a	0.870±0.048^a
TSA+miR-4298 inhibitor组	0.386±0.025^b	0.570±0.036^a
F值	68.729	121.259
P值	<0.001	<0.001

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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

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