卫生研究

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(0,10,20,30,40 μmol/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(CdCl₂, 20 μmol/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 of DDIT4 in the cadmium+miR-221-3p mimic group was down-regulated(P<0.05), and the ratio of Bcl-2/BAX was increased from 0.54±0.03 to 0.71±0.04.CONCLUSION miR-221-3p inhibits cadmium-induced apoptosis of TM3 cells by targeting DDIT4.