LncRNAWSF27/miR-1696 Contributes To Selenium Deficiency-induced Lymphocyte Necroptosis By Targeting Gpx3 In Chicken Spleen

Selenium(Se),as an essential micronutrient,plays a vital role in maintaining the homeostasis and normal life activities both in human and animals.The existence of Se is indispensable for human and animals to maintain the normal physiological function and process of tissues and organs.The main functions of Se are anti-oxidation and anti-inflammatory.Se deficiency can induce various injuries of tissues and organs,including brains,hearts,livers,pancreas,muscles,thymus,spleens and even death in chickens.Therefore,Se deficiency disease,as one of the nutritional metabolic diseases,will cause huge economic loss in the poultry industry.Selenoprotein is the main form and carrier for Se to play its biological functions in animals,and the major function of selenoproteins is antioxidation.Gpx3 is an important member of glutathione peroxidase(Gpxs)family,and has significant antioxidant funtion.Some studies pointed out that the development,differentiation and other cellular process would be influenced by Gpx3 level.Moreover,when the level of Gpx3 decreased,the ability of antioxidant reduced greatly,and immune functions weakened by a large margin.Besides,several researches indicated that Gpx3 has the function of anti-oxidation and anti-inflammatory in poultry,and participates in the immune tissue damage caused by Se deficiency,but its specific biological function in broiler has not been determined.Spleen inflammation and diseases are intricately connected to lymphocyte cell death,and the main form of lymphocyte cell death induced by Se deficiency is necrosis.Necroptosis,a kind of cell death,is procedural and non-apotosis.It has been reported that necroptosis plays a key role in immune dysfunction,spleen disease and inflammatory process.And it was confirmed to have close relationship with tissue and organ injuries and other diseases induced by Se deficiency.Micro RNA(miRNA),does not have the function of translation proteins,and it has been demonstrated to participate in multiple biological reactions by regulating post-transcriptional expression of its target genes,and acts a pivotal part in regulating programmed cell death.At present,it has not been reported lncRNA is involved in spleen injury and lymphocyte programmed necrosis induced by Se deficiency in broilers.Consequently,in the present study,the broiler was used as the research object,Se-deficient diet(0.008 mg/kg Se)was fed to establish the model of exudative quality broilers in vivo.Establishment of knockdown/overexpression model of Gpx3,miR-1696 and lncRNAWSF27 in chicken spleen lymphocytes by in vitro transfection.By using H.E.staining,transmission electron microscopy,fluorescence microscopy,lncRNA/mRNA omics and RNA-seq,bioinformatics analysis,western blot,quantitative Real-time PCR(qRT-PCR),flow cytometry,AO/EB staining,spleen histomorphological changes,lncRNA expression profiles,necroptosis were detected,as well as analysis of the signal transduction pathways related to necroptosis,and construction and validation of lncRNA-miRNA-mRNA regulatory network.The aim and target of presentstudyis to investigate the function and effects of Gpx3 in the programmed necrosis of spleen lymphocytes induced by Se deficiency and to elucidate the mechanism of lncRNAWSF27 and miR-1696 regulating the programmed necrosis of lymphocytes by targeting Gpx3.The results showed that:(1)Exudative quality model of broiler was established successfully by feeding Se-deficient diet.Histopathological observation of broiler spleens revealed that the number and volume of lymphocytes decreased,the distribution of arterial lymphoid sheath was loose,the space between splenic sinuses was enlarged,and the red pulp was congested in the exudative quality chicken spleens,in addition,lymphonecrosis and cell edema were observed in spleen of broilers in Se deficiency group.Transmission electron microscopy observation showed that Se deficiency could lead to cell membrane and nuclear membrane destruction,nuclear pyknosis and nuclear fragmentation.The above-mentioned was typical characters of necrosis,and the results of qRT-PCR and western blot showed that Se deficiency could activate MAPK signaling pathway(Erk,JNK and p38)phosphorylation,increase the expressions of IL-1β,IL-6,IL-8,IL-17,TNF-α,NF-κB,i NOS,COX-2,PTGE,and necrosis related genes(RIPK1,RIPK3,MLKL),decrease the expression of Caspase8.Theses results suggested Se deficiency could activate MAPK/NF-κB signaling pathway and induce necrosis in chicken spleen.(2)According to the results of transcriptome detection,it showed that low Se deficiency could cause differential expression of 21 lncRNAs and 337 mRNAs.Among them 12 lncRNAs and 210 mRNA increased while 9 lncRNAs and 127 mRNAs decreased(including lncRNAWSF27 and Gpx3)(log2 fold change > 0.585 or <-0.585,FDR < 0.05).The results of enrichment analysis showed proliferation,differentiation and death of immune cells,immune response,cell death and clearance,and nutrition metabolism were affected.(3)Bioinformatics analysis predicted that lncRNAWSF27 could bind to miR-1696 using the same response element site of Gpx3,and the expressions of lncRNAWSF27 and Gpx3 increased and the expression of miR-1696 decreased in Se deficiency broiler spleen.It suggested lncRNAWSF27,miR-1696 and Gpx3 had potential interaction.The targeting relationships between lncRNAWSF27 and miR-1696,miR-1696 and Gpx3 were confirmed by dual-luciferase reporter assay.In addition,knockdown of miR-1696 would increase the expression of Gpx3 and overexpression of miR-1696 would inhibit the expression of Gpx3;knockdown lncRNAWSF27 could increase miR-1696 and decrease Gpx3 level,and overexpression of lncRNAWSF27 could inhibit miR-1696 level and increase the expression of Gpx3 level in vitro cultured spleen lymphocytes.These results indicated that lncRNAWSF27 can release the inhibitory effect of miR-1696 on Gpx3 by acting as miR-1696 sponge.(4)After establishing the models of overexpression/knockdown of lncRNAWSF27,miR-1696 and Gpx3,the expression of programmed necrosis related genes were detected.The results showed low levels of lncRNAWSF27 and Gpx3 or high level of miR-1696 could induce the expressions of RIPK1,RIPK3 and MLKL increased significantly,and the levels of Caspase8 showing the opposite trend.In addition,overexpression of lncRNAWSF27 and Gpx3 or knockdown of miR-1696 also caused activities of SOD,T-AOC,GSH decline,levels of H2O2 and ROS increased markedly,and MAPK signaling pathway activated,the expressions of inflammatory factors increased.These results suggested that the down-regulation of lncRNAWSF27 and Gpx3 or up-regulation of miR-1696 could induce oxidative stress,increase ROS levels,activate MAPK/NF-κB pathways and lead to programmed necrosis occurred in lymphocytes.(5)Before establishing the models of programmed necrosis lymphocytes by using programmed necrosis activator z-VAD-fmk,lncRNAWSF27 and Gpx3 were overexpression,miR-1696 was knockdown through transfection,respectively.We found that the ROS levels decreased significantly,the expressions of MAPK/NF-κB signaling pathways declined,and programmed necrosis was inhibited,compared with the programmed necrosis model(z-VAD-fmk),and the results of AO/EB staining showed the high levels of lncRNAWSF27 and Gpx3,or low level of miR-1696 could decreased programmed necrosis markedly.These results indicated lncRNAWSF27 and Gpx3 protected the life of lymphocytes from programmed necrosis caused by high expression of miR-1696.In summary,Se deficiency could cause oxidative stress and necroptosis in chicken spleen,and alter lncRNA and mRNA expression profile(12 lncRNAs and 210 mRNA increased while 9lncRNAs and 127 mRNAs decreased).The expression of lncRNAWSF27 was decreased,as the expression of miR-1696 increased and the expression of Gpx3 decreased,which led to spleen tissue oxidative stress,inflammation and cell necroptosis.In vitro,we confirmed that Gpx3 is a specific target gene downstream of miR-1696,and its expression is regulated by miR-1696;and lncRNAWSF27 can specifically bind to miR-1696 and weaken its inhibitory effect on Gpx3 as a molecular sponge.Further studies showed that miR-1696 could inhibit the expression of Gpx3,increase the ROS content of lymphocytes,and lead to oxidative stress,inflammatory response and programmed necrosis;and overexpression of lncRNAWSF27 could alleviate these processes and protect lymphocytes.The present study revealed a new regulatory mechanism of spleen nerosis,which was composed of lncRNAWSF27,miR-1696,Gpx3 and dietary Se content.In other words,Se deficiency can induce oxidative stress by regulating lncRNAWSF27/miR-1696/Gpx3 axis,activate MAPK/NF-κB signaling pathway,and cause programmed necroptosis of spleen lymphocytes in broiler.This conclusion not only enriches the understanding of the mechanism of spleen injury and diseases caused by nutritional metabolism,but also provides possible preventive and therapeutic basis for Se deficicncy-related diseases,and provide decision-making basis for healthy farming.