Regulatory effect of 5'tRF-LysCTT on ferroptosis in mouse cardiomyocytes and its mechanism

Abstract

Objective To investigate the regulatory effect of tRNA-derived small RNA (tsRNA) on ferroptosis in mouse cardiomyocytes and its mechanism. Methods RT-qPCR was used to measure the relative expression level of 5'tRF-LysCTT in the heart, liver, spleen, kidney, brain, and muscle of C57BL/6J mice aged 8 weeks. Primary cultured cardiomyocytes were divi-ded into groups A, B, C, and D: the cardiomyocytes in group A were cultured in a complete medium for 60 h; those in group B were cultured in a complete medium for 24 h, followed by starvation treatment for 12 h and H/R treatment for 24 h; those in groups C and D were transfected with antagomir-NC and antagomir-5'tRF-LysCTT, respectively, and were given starvation treatment (12 h) and H/R treatment (12 h) after 24 h of transfection. Primary cultured cardiomyocytes were divided into groups E, F, and G: the cardiomyocytes in group E were cultured in a complete medium for 24 h; those in group F were transfected with agomir-NC for 24 h; those in group G were transfected with agomir-5'tRF-LysCTT for 24 h. RT-qPCR was used to measure the relative expression levels of 5'tRF-LysCTT, Ptgs2, Gpx4, and Slc7a11 in mouse cardiomyocytes of groups A-G; ferrous ion colorimetric test kit, lipid reactive oxygen species (ROS) staining, and CCK8 assay kit were used to measure the relative content of ferrous ions, ROS level, and the survival rate of cardiomyocytes in groups A-G; Prussian blue staining was used to observe iron ion deposition in cardiomyocytes. Results RT-qPCR showed that the expression level of 5'tRF-LysCTT in heart was significantly higher than that in the liver, spleen, kidney, brain, and muscle (F=16.21,t=3.81-7.93,P<0.05). Compared with group B, group D had significant reductions in the relative expression levels of 5'tRF-LysCTT and Ptgs2, significant increases in the relative expression levels of Gpx4 and Slc7a11, significant reductions in the relative content of ferrous ions and ROS level, and a significant increase in the survival rate of cardiomyocytes (t=3.26-15.61,P<0.05), as well as a significant increase in iron ion deposition in cardiomyocytes, while there were no significant differences in the above indicators between group C and group B (P>0.05). Compared with group E, group G had significant increases in the relative expression levels of 5'tRF-LysCTT and Ptgs2, significant reductions in the relative expression levels of Gpx4 and Slc7a11, significant increases in the relative content of ferrous ions and ROS level, and a significant reduction in the survival rate of cardiomyocytes (t=3.57-91.84,P<0.05), as well as a significant reduction in iron ion deposition in cardiomyocytes, while there were no significant differences in the above indicators between group F and group E (P>0.05). Conclusion 5'tRF-LysCTT can regulate ferroptosis in mouse cardiomyocytes, and knockdown of 5'tRF-LysCTT can inhibit cardiomyocyte ferroptosis induced by H/R, while overexpression of 5'tRF-LysCTT can promote cardiomyocyte ferroptosis.

Keywords

• rna, transfer|rna, small untranslated|hypoxia|myocardial reperfusion injury|myocytes, cardiac|ferroptosis