Mechanism of miR-221-3p inhibiting cadmium-induced apoptosis of TM3 cells through DDIT4
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.
NETL
NSTL
万方数据
