| Selenium(Se)is an important micronutrient.Selenium deficiency can cause many diseases,such as white myopathy,liver necrosis and ferroptosis.Ferroptosis is a newly discovered programmed cell death characterized by iron-dependent accumulation of peroxides,which is a novel cell death pattern distinct from typical apoptosis and other programmed cell death.Due to selenium deficiency,the selenoprotein GPX4 is reduced and the lipid reactive oxygen species(ROS)levels are affected,leading to the occurrence of ferroptosis.Micro RNA is a class of highly conserved noncoding RNA,which regulates the expression of corresponding proteins by binding to the 3’UTR region of target genes,and plays an important role in the regulation of ferroptosis in cells.Studies have found that the expression of various micro RNA in tissues changes significantly when the selenium content in the body is decreased.In this study,the selenium deficient broiler model was replicated to detect the pathological structure of liver and the expression level of ferroptosis gene,and the mi R-129-3p with selenium deficiency specificity was screened out.In combination with related bioinformatics software,the target genes and possible signal transduction pathways were predicted and verified.The specific target genes of mi R-129-3p were analyzed and verified by dual luciferase reporter assay system,q RT-PCR and Western Blot,and the mechanism of action of mi R-129-3p on ferroptosis in liver cells of selenium deficient broilers was studied.The main research results are as follows:(1)After collecting the liver tissues of Se deficient broilers in this study,the microscopic structure of the liver tissues was observed by fixation and staining.It was found that the liver tissues of selenium deficient broilers showed disordered liver cells,bleeding,increased cell space,inflammatory cell infiltration and adipocyte degeneration.(2)Mi R-129-3p is one of the selenium deficiency specific micro RNAs,which can induce the overexpression of mi R-129-3p in broiler liver tissue cells.Target gene prediction was carried out through mi RDB,mi RBase and other websites to screen out potential mir-129-3p target gene SLC7A11,and target gene verification was carried out through dual luciferase reporter gene method.Then,by building the knockdown/overexpression model of mi R-129-3p in the subcultured broilers liver cancer cells,the gene and protein expressions of SLC7A11 were detected by Western Blot and QRT-PCR.The results showed that the m RNA and protein levels of SLC7A11 were significantly decreased in the overexpression group,while significantly increased in the knockdown group.SLC7A11 was confirmed again as the target gene of mir-129-3p.(3)In this study,Western Blot and q RT-PCR detection showed that low-selenium feed could significantly up-regulate the expression of mi R-129-3p and down-regulate the expression of SLC7A11,down-regulate the expressions of genes related to SLC7A11/GPX4 pathway,and significantly up-regulate the expressions of iron TFR1 and DMT1 in the liver tissue of broilers.Keap1 expression in oxidative stress pathway was significantly increased,while Nrf2 and HO-1expression were significantly down-regulated.These results indicate that mi R-129-3p can activate genes related to ferroptosis pathway and induce oxidative stress in broiler liver cells by targeting SLC7A11.(4)Based on the overexpression/inhibition model of mi R-129-3P in LMH cells,the cells were treated with Erastin and ferrostatin-1.The m RNA and protein levels of genes related to ferroptosis pathway were detected by Western Blot and q RT-PCR,and the results showed that mi R-129-3p could induce ferroptosis in broilers liver cancer cells by inhibiting the expression of SLC7A11.In summary,when broilers are deficient in selenium,the expression of mi R-129-3p increases,and by targeting SLC7A11 to induce ferroptosis in hepatocytes,the above information provides new data for understanding the mechanism of liver damage caused by selenium deficiency. |
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