| Selenium is an essential trace element in organisms.Many diseases are related to insufficient intake of selenium,such as Keshan disease,Kashin-Beck disease,certain tumors,diabetes,hypertension,immune and reproductive dysfunction.In livestock and poultry breeding,selenium deficiency in poultry mainly manifests as white muscle disease,exudative quality,usually with neurological symptoms,slow growth and decreased egg production rate.The liver is also one of the main target organs for selenium deficiency diseases.Selenium deficiency cause liver necrosis.Transcriptome analysis aims to capture coding and non-coding RNA（nc RNA）,and quantify the changes of gene expression in cells,tissues,organs,and even the entire body.It provides a basis for exploring the regulation pathways and genetic networks of phenotypes,and is important to agriculture and human medicine.Micro RNA（miRNA）,as a kind of nc RNA,mediate a variety of biological processes by targeting and regulating its target gene at the post-transcriptional levels,and play an important regulatory role in the occurrence of various diseases.In recent years,studies found that the expression level of miRNA also responds to the selenium content in the body,and participates in various tissue damages caused by selenium deficiency.Long non-coding RNA（lncRNA）can combine with miRNA in the cytoplasm and function as a molecular sponge to regulate miRNA.At present,whether lncRNA is involved in liver damage induced by selenium deficiency,and its function has not been reported.In this study,broiler chickens were used as the research object.The selenium-deficient liver necrosis model of chickens was established by feeding selenium-deficient diets with a selenium content of 0.008 mg/kg.H&E staining,transcriptomics detection technology,real-time fluorescent quantitative PCR,immunoblotting,bioinformatics methods were applied to detect liver tissue morphological changes,mRNA/lncRNA/miRNA expression profiles,necroptosis associated signal transduction pathways,construct and verify lncRNA-miRNA-mRNA regulatory network.Subsequently,the selected specific lncRNA and miRNA were knocked down and/or overexpressed in chicken LMH cells in vitro,and analyzed regulation of its target genes and downstream pathways,as well as the impact on the occurrence of necroptosis.The main results are as follows:（1）Histopathological results showed that liver cell morphology was irregular,nucleus was concentrated,and hepatocyte necrosis in chicken livers after feeding low-selenium diets,indicating that selenium deficiency induced necrotic lesions of chicken liver tissue.（2）Selenium deficiency caused up-regulation of 390 mRNAs and 40 lncRNAs,down-regulation of 245 mRNAs,49 lncRNAs and 5 miRNAs（log2 fold change>0.585 or<-0.585,FDR<0.05）in chicken liver tissues,the enrichment analysis found that the metabolism and redox process were seriously affected.（3）Bioinformatics analysis found a potential interaction relationship between lncRNA Tcons＿18040481/miR-7b/TNFR1,and the dual luciferase reporter analysis test confirmed that the binding sites were effective.The location of cytoplasm and nucleus showed that Tcons＿18040481 was mainly located in the cytoplasm.In the liver of selenium-deficient chickens,the expression of Tcons＿18040481 and TNFR1 was up-regulated,while the expression of miR-7b was down-regulated.In addition,overexpression or knockdown of miR-7b in LMH cells could inhibit or increase the expression of TNFR1 at mRNA and protein levels,respectively.Overexpression of Tcons＿18040481 reduced the expression of miR-7b in LMH cells,and increased the protein and mRNA levels of TNFR1;accordingly,knocking down Tcons＿18040481 increased the level of miR-7b and inhibited the expression of TNFR1.It suggests that Tcons＿18040481 can act as a molecular sponge of miR-7b to regulate the expression of miR-7b and target gene TNFR1.（4）The protein detection results of selenium-deficient chicken liver showed that the expression levels of TNFα,TNFR1,RIP1,RIP3 and MLKL were up-regulated,the protein expression of caspase 8 was decreased,indicating that selenium deficiency caused chicken hepatocyte necroptosis.In LMH cells,miR-7b inhibition resulted in the increase of cell necrosis and expression of RIP1,RIP3,and MLKL,which could be alleviated by Nec-1 treatment,indicating that low miR-7b expression induced RIP-dependent necroptosis in chicken hepatocytes.When miR-7b was inhibited and TNFR1 was knocked down using si RNA together,it was found that TNFR1 inhibition could alleviate the levels of necroptosis caused by the low miR-7b.Combined with the targeted regulation relationship between miR-7b and TNFR1,it showed that miR-7b suppression caused necroptosis by releasing TNFR1.In addition,overexpressing miR-7b alleviated the necroptosis of LMH cells caused by z VAD-fmk and TNFαco-treatment,indicating that miR-7b could block necroptosis by inhibiting TNFR1.The exploration of Tcons＿18040481 found that overexpressing Tcons＿18040481 can cause the occurrence of necroptosis in LMH cells,which can also be alleviated by Nec-1 and TNFR1inhibition.The above results indicate that Tcons＿18040481 acts as the ce RNA of miR-7b to increase the expression of TNFR1 and participates in the necroptosis of hepatocytes caused by selenium deficiency.（5）Selenium deficiency induced increased protein expression levels of TNFα,TNFR1,IKKα/β,IκBαand NF-κB in chicken liver tissues.In vitro results showed that Tcons＿18040481overexpression and miR-7b inhibition increased the protein expression of IKKα/β,IκBαand NF-κB,and the mRNA expression of pro-inflammatory cytokines i NOS,COX-2,PGE and TNF,indicating that Tcons＿18040481/miR-7b activated the NF-κB pathway.Inhibition of TNFR1alleviates the upregulation of NF-κB and TNFαinduced by Tcons＿18040481 overexpression and miR-7b inhibition,which claimed that Tcons＿18040481/miR-7b increased TNFR1 expression and activated the NF-κB pathway in selenium-deficient chicken liver.（6）In vitro cultured LMH cells,antioxidant NAC treatment reversed miR-7b inhibition and Tcons＿18040481 induced hepatocyte necrosis in chickens,indicating that oxidative stress contributed to Tcons＿18040481/miR-7b induced necroptosis.In addition,inhibition of miR-7b and overexpression of Tcons＿18040481 resulted in increased H2O2and ROS level,enhanced activities of SOD and GSH,along with activation of the NF-κB pathway and necroptosis.These changes were inhibited by antioxidant NAC or necroptosis inhibitor Nec-1,suggesting enhanced intracellular antioxidant function in response to oxidative stress.NAC treatment reversed the activation of NF-κB and TNFαinduced by miR-7b inhibition,confirming that oxidative stress and inflammatory response induced by Tcons＿18040481/miR-7b promoted each other.Tcons＿18040481/miR-7b is also involved in oxidative stress caused by selenium deficiency in combination with transcriptomics in chicken livers,which showing that the redox process is affected during selenium deficiency.In summary,these results indicate that Tcons＿18040481/miR-7b axis increases TNFR1 expression,thereby induced oxidative stress and caused hepatocyte necroptosis in the liver of selenium-deficient chickens.To sum up,Se deficiency caused RIP-dependent necroptosis in chicken liver tissue,induced differential expression of 635 mRNAs,5 miRNAs,and 89 lncRNAs.TCONS＿18040481 could regulate the expression of miR-7b target gene TNFR1.Further research confirmed that Se deficiency leads to increased TCONS＿18040481 expression in chicken liver,by inhibiting miR-7b expression,to release TNFR1 and activate NF-κB signal,and cause oxidative stress,causing necroptosis of chicken hepatocytes.The results indicated that Se deficiency regulated TCONS＿18040481/miR-7b/TNFR1 pathway,activated NF-κB/ROS signaling pathway,induced necroptosis of chicken hepatocytes.The results of this study provide a reference for further research,providing possible prevention and treatment strategies for necroptosis-induced liver injury,At the same time,it provides a decision basis for the healthy breeding of livestock and poultry. |
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