微小RNA-221-3p通过DDIT4抑制镉诱导的TM3细胞凋亡机制  

Mechanism of miR-221-3p inhibiting cadmium-induced apoptosis of TM3 cells through DDIT4

作　　者：罗皓珑 陈梦圆 骈雅婧 周丽 任香梅 Luo Haolong;Chen Mengyuan;Pian Yajing;Zhou Li;Ren Xiangmei(Department of Nutrition,School of Public Health,Xuzhou Medical University,Key Laboratory of Environment and Health of Xuzhou,Xuzhou 221004,China)

机构地区：[1]徐州医科大学公共卫生学院营养学教研室,徐州市环境与健康重点实验室,徐州221004

出　　处：《卫生研究》2024年第3期478-486,共9页Journal of Hygiene Research

基　　金：国家自然科学基金(No.81302429);江苏省研究生科研与实践创新计划项目(No.KYCX21_2726)。

摘　　要：目的探讨微小RNA(microRNA,miRNA)中的miR-221-3p靶向DNA损伤诱导转录本4(DNA-damage-inducible transcript 4,DDIT4)对镉诱导小鼠睾丸间质细胞凋亡的影响和机制。方法小鼠睾丸间质细胞(mouse testicular stromal cells,TM3)经不同浓度的镉(0、10、20、30和40μmol/L)染毒后,使用CCK-8检测细胞活性。提取镉染毒TM3细胞的总RNA,以差异倍数(fold change,FC)>1.2,P<0.05作为标准筛选显著差异表达miRNA。TM3细胞分为空白对照组、阴性对照组、镉染毒组(CdCl2,20μmol/L)和镉染毒+miR-221-3p模拟物组,其中对镉染毒+miR-221-3p模拟物组,先将miR-221-3p模拟物转染进TM3细胞,再联合镉染毒24 h。Hoechst染色检测细胞形态,流式细胞仪分析细胞凋亡率,实时荧光定量PCR和Western blot免疫印迹法检测DDIT4表达水平,双荧光素酶报告基因实验验证miR-221-3p与DDIT4的结合,生物信息学分析DDIT4的功能及与细胞凋亡的关联,过表达miR-221-3p后观察B淋巴细胞瘤-2(B-cell lymphoma-2,Bcl-2)和B淋巴细胞瘤-2相关X蛋白(Bcl-2 associated X protein,BAX)的表达水平。结果镉染毒可降低TM3细胞活性且存在剂量-效应关系。细胞形态学发现,与对照组相比,镉染毒组细胞皱缩、细胞核浓染,凋亡率升至19.66%±0.45%(P<0.01);与镉染毒组相比,镉染毒+miR-221-3p模拟物组正常形态细胞增多,凋亡率降至13.76%±0.37%(P<0.05)。镉染毒组miR-221-3p表达水平下调(P<0.01),DDIT4表达水平上调(P<0.05)。生物信息学分析和双荧光素酶报告分析发现DDIT4为miR-221-3p的靶基因之一。与镉染毒组相比,镉染毒+miR-221-3p模拟物组DDIT4表达水平下调(P<0.05),Bcl-2/BAX比值由0.54±0.03增加为0.71±0.04。结论miR-221-3p通过靶向DDIT4进而抑制镉诱导的TM3细胞凋亡。OBJECTIVE To investigate the mechanism of DNA-damage-inducible transcript 4(DDIT4)targeting miR-221-3p in microRNA(miRNA)on cadmium-induced apoptosis of mouse testicular stromal cells.METHODS The activity of mouse testicular interstitial cells(TM3)was detected by CCK-8 after exposure to different concentrations of cadmium(0,10,20,30,40μmol/L).Total RNA was extracted from cadmium-treated TM3 cells,and the significantly differentially expressed miRNA was screened with fold change(FC)>1.2 and P<0.05 as the criterion.TM3 cells were divided into blank control group,negative control group,cadmium exposure group(CdCl_2,20μmol/L),and cadmium+miR-221-3p mimic group.miR-221-3p mimic group was transfected into TM3 cells first,combined with cadmium exposure for 24 hours.The cell morphology was detected by Hoechst staining,and the apoptosis rate was analyzed by flow cytometry.Quantitative real-time PCR(qRT-PCR)and Western blot were used to detect DDIT4 expression.Dual luciferase reporter gene assay verified the binding of miR-221-3p to DDIT4.The function of DDIT4 and its relationship with apoptosis were analyzed by bioinformatics.The expression levels of B-cell lymphoma-2(Bcl-2)and Bcl-2 associated X protein(BAX)were observed after overexpression of miR-221-3p.RESULTS Cadmium treatment of TM3 cells could reduce cell activity and there was a dose-effect relationship.The cell morphology showed that compared with the control group,the cells were wrinkled and the nuclei were heavily stained,and the apoptosis rate increased to 19.66%±0.45%(P<0.01).Compared with the cadmium exposure group,the normal morphologic cells increased in the cadmium exposure+miR-221-3p mimic group,and the apoptosis rate decreased to 13.76%±0.37%(P<0.05).The expression level of miR-221-3p was down-regulated(P<0.01),and the expression level of DDIT4 was up-regulated(P<0.05).Bioinformatics analysis and dual luciferase report analysis showed that DDIT4 was one of the target genes of miR-221-3p.Compared with the cadmium exposure group,the expression level o

关 键 词：镉 细胞凋亡 微小RNA-221-3p DNA损伤诱导转录本4 

分 类 号：R114[医药卫生—卫生毒理学] R12[医药卫生—公共卫生与预防医学]