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

Abstract

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. 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(CdCl_2, 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. 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. miR-221-3p inhibits cadmium-induced apoptosis of TM3 cells by targeting DDIT4.