The Role Of LncRNA-TCONS-6251/miR-novel-100/TC2N Axis Mediated ROS Regulation Of Ferroptosis In The Duck Kidney Injury Induced By Copper

The aim of this study is to investigate the regulatory mechanism on reactive oxygen species（ROS）and ferroptosis of lncRNAs-related ceRNA induced by copper（Cu）in duck kidney.Peking ducks were divided into a control group（10 mg/kg）and a Cu group（400 mg/kg）,and fed with a Cu-containing diet for 45 days continuously.Subsequently,transcriptome sequencing was performed on the kidney tissue of Peking ducks to obtain the differentially expressed lncRNAs and related ceRNA networks.In addition,the ferroptosis-related factors in duck kidney were determined by western blotting and real-time quantitative PCR.To verify the effect of Cu exposure on ferroptosis in vitro,a model of duck renal tubular epithelial cells exposed to Cu was established.The dual-luciferase reporter gene system was used to verify the targeting relationship between miR-novel-100 and lncRNA-TCONS-6251 or TC2N.Subsequently,the effects of miR-novel-100 overexpression or knockdown and TC2N knockdown on ROS levels,changes in the morphology of ferroptotic cells and the levels of ferroptosis-related indicators were determined.After Cu exposure,the expressions of lncRNA-TCONS-6251 and TC2N were down-regulated,while the miR-novel-100 was up-regulated（P<0.05）.The results of the dual luciferase gene reporter system proved that miR-novel-100 was targeted with lncRNA-TCONS-6251 and TC2N.After knockdown or overexpression of the miR-novel-100,the expression trends of TC2N and lncRNA-TCONS-6251 were negatively correlated with miR-novel-100.After TC2N knockdown,the expression of lncRNA-TCONS-6251 was down-regulated,and the expression level of miR-novel-100 was up-regulated.The transmission electron microscopy results showed that the outer mitochondria were ruptured and the mitochondrial cristae were blurred caused by Cu in vitro and in vivo.Immunofluorescence observation found that the level of lipid peroxide was increased in the Cu group（P<0.01）.After Cu treatment,GSH level was decreased,MDA and Fe²⁺levels were increased,upregulated the ferroptosis-related genes（NRF2,ACSL4,PTGS2 and TFR1）expression levels（P<0.05）,and downregulated the ferroptosis-related genes（GPX4,SLC7A11 and FTH1）expression levels（P<0.05）.Knockdown of miR-novel-100 could suppress the level of Cu-induced ferroptosis.Compared with the Cu group,knockdown of miR-novel-100 down-regulated the levels of ferroptosis-related indicators and up-regulated the expressions of lncRNA-TCONS-6251 and TC2N.In conclusion,the results suggest that the ferroptosis induced by Cu via the lncRNA-TCONS-6251/miR-novel-100 axis.Knockdown of TC2N or overexpression of miR-novel-100 caused the ROS accumulation and up-regulation of ferroptosis indicators（P<0.05）.In addition,the up-regulation of ferroptosis indicators and ROS accumulation caused by miR-novel-100overexpression were decreased after the butyl hydroxy anisole（BHA）treatment.The promoting effect of TC2N knockdown on ferroptosis and ROS accumulation were inhibited induced by miR-novel-100 knockdown.The results showed that miR-novel-100/TC2N axis induced ferroptosis via ROS accumulation.In summary,Cu could regulate ROS level to mediate ferroptosis through the lncRNA-TCONS-6251/miR-novel-100/TC2N axis in duck kidney.