LncRNA3037/miR-15a Contributes To H₂S-induced Apoptosis And Necroptosis By Targeting BCL2 And A20 In Chicken Trachea

Hydrogen sulfide（H₂S）is a dangerous air pollutant.With the development of industrialization,the hazards caused by H₂S pollution in the air gradually attract people’s attention.H₂S has toxic effects on the tissues and organs of the respiratory system,cardiovascular system and nervous system in organisms.Apoptosis and necroptosis are the main forms of exogenous toxicant-induced cell death,and play an important role in respiratory dise ases.Micro RNA（mi RNA）,as a kind of non-coding RNA,participates in multiple biological reactions by regulating post-transcriptional expression of its target genes,and plays an important role in regulating programmed cell death.In recent years,studies show that mi RNAs play a major role in the regulation of some environmental pollutant poisoning and diseases.mi RNAs are gradually recognized as potential biomarkers for the connection of environmental pollutants to certain diseases.Long non-coding RNAs（lnc RNAs）bind to mi RNAs in the cytoplasm and act as mi RNAs sponge to regulate the expression of mi RNAs.At present,whether lnc RNA is involved in H₂S-induced respiratory tract damage,and its function has not been reported.Therefore,in this experiment,the broiler was used as the research object,and H 2S gas was continuously introduced into the environmentally controlled chamber to establish the model of H₂S exposure chicken in vivo.Na HS,the donor of H₂S,was used to establish the model of H₂S exposure chicken LMH cells in vitro.By using transmission electron microscopy,scanning electron microscopy,RNA-seq,western blot,quantitative Real-time PCR,flow cytometry,bioinformatics analysis,tracheal histomorphological changes,lnc RNA expression profiles,apoptosis and necroptosis were detected,as well as analysis of the signal transduction pathways related to apoptosis and necroptosis,and construction and validation of lnc RNA-mi RNA-m RNA regulatory network.The study aims to prove the type of cell death in chicken tracheal caused by H₂S exposure,and the role of lnc RNA during H₂S exposure.The results showed that:（1）Histopathological observation of chicken trachea revealed that H₂S exposure caused epithelial cells shed,ciliated cells severely deficient,and mass inflammatory cells infiltrated in the lesion.Scanning electron microscopy revealed that the tracheal epithelial cilia were missing and messy,and a large amount of mucus adhered to the surface of the cilia.Transmission electron microscopy observation showed that H₂S exposure could lead to cell membrane and nuclear membrane destruction,chromatin condensation and edge aggregation,mitochondrial swelling,rupture and vacuolization,rough endoplasmic reticulum degranulation and fracture.The above-mentioned was typical characters of necrosis and apoptosis,which demonstrated that H₂S exposure caused necrosis and apoptosis in chicken trachea.（2）H₂S exposure caused up-regulation of 6 lnc RNAs and down-regulation of 10 lnc RNAs（FDR < 0.05,absolute fold-change > 1.5）in chicken tracheal.The verification of partial transcripts by q RT-PCR confirmed the reliability of transcription sequencing.It indicated that H₂S exposure can affect the expression of lnc RNA in chicken tracheal tissue.In addition,intracellular localization of the five down-regulated lnc RNAs in the H₂S group revealed that lnc RNA3037 is the most down-regulated lnc RNA in the cytoplasm.（3）Bioinformatics analysis predicted that lnc RNA3037 could bind to mi R-15 a,and H₂S exposure increased the expression of mi R-15 a in chicken tracheal,suggesting that lnc RNA3037 might act as mi R-15 a sponge.Dual-luciferase reporter assay confirmed that lnc RNA3037 was able to bind mi R-15 a.Overexpression and knockdown of lnc RNA3037 and mi R-15 a in chicken LMH cells in vitro confirmed the relationship of negatively regulation between lnc RNA3037 and mi R-15 a.In addition,knockdown of lnc RNA3037 or overexpression of mi R-15 a inhibited the the expression of BCL2 and A20,and knockdown of mi R-15 a reversed the inhibitory effect of lnc RNA3037 on BCL2 and A20.These results indicated that lnc RNA3037 can regulate the expression of the target genes A20 and BCL2 of mi R-15 a by acting as mi R-15 a sponge.（4）H₂S exposure increased the expression of necroptosis related proteins RIPK1,RIPK3 and p-MLKL,decreased the expression of anti-necroptosis factor A20,increased the expression apoptosis-inducing factors BAX and caspase 3,and decreased the expression of anti-apoptotic factor BCL2 in tracheal tissues.The experiments results in vitro showed that the activity of LMH cells decreased in a concentration-dependent manner after H₂S exposure,and the apoptosis and necrosis rate increased,necroptosis-and apoptosis-related proteins also had corresponding expression patterns.It was confirmed that H₂S exposure caused necroptosis and apoptosis in chicken tracheal and LMH cells.On the basis of the model of mi R-15 a overexpression/knockdown LMH cells,cells were treated with necroptosis inhibitor（Nec-1）,apoptosis inhibitor（z-VAD-fmk）,Na HS and si RNA-lnc RNA3037,the results showed that overexpression of mi R-15 a could induce apoptosis and necroptosis.Pretreatment with Nec-1 or z-VAD-fmk,respectively inhibited the necroptosis and apoptotic rate induced by overexpression of mi R-15 a.In addition,knockdown of mi R-15 a reduced Na HS-and knockdown of lnc RNA3037-induced apoptosis and necroptosis.These results indicated that H₂S exposure-induced mi R-15 a could cause apoptosis and necroptosis by inhibiting the expression of its target genes BCL2 and A20.In summary,H₂S exposure could cause inflammatory injury,apoptosis and necroptosis in chicken tracheal,and induce changes in lnc RNA expression profile.The expression of lnc RNA3037,which located in the cytoplasm was decreased,as the expression of mi R-15 a increased and expression of A20 and BCL2 decreased.It confirmed that A20 and BCL2 were the target genes of mi R-15 a,and lnc RNA3037 could bind to mi R-15 a and regulate the expression ofBCL2 and A20.Further studies showed that inhibition of lnc RNA3037 expression could down-regulate the expression of BCL2 and A20 by regulating mi R-15 a,leading to apoptosis and necroptosis.The results of this study not only provide scientific basis for H 2S toxicology research,but also provide possible preventive and therapeutic basis for H₂S exposure-related diseases,and provide decision-making basis for healthy farming.