Regulatory effect of 5'tRF-LysCTT on ferroptosis in mouse cardiomyocytes and its mechanism
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 treat-ment(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 colorimet-ric 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 depo-sition 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 expres-sion levels of Gpx4 and Slc7a11,significant reductions in the relative content of ferrous ions and ROS level,and a significant in-crease 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 car-diomyocytes,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 ex-pression levels of 5'tRF-LysCTT and Ptgs2,significant reductions in the relative expression levels of Gpx4 and Slc7a11,signifi-cant 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 signifi-cant differences in the above indicators between group F and group E(P＞0.05).Conclusion 5'tRF-LysCTT can regulate fer-roptosis 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.
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