Screening Of Inflammation-related LncRNAs In Bovine Mammary Epithelial Cells And Function Study Of Key LncRNA

lncRNA,as an endogenous non-coding RNA,can regulate several biological processes such as immune system and biological development.In this study,LPS-induced bovine mammary epithelial cells（bMECs）were used as the study material,and RNA-seq was performed by Illumina PE 150 to screen differentially expressed lncRNAs and mRNAs.The expression pattern of some differentially expressed lncRNAs was detected by qRT-PCR,the subcellular localization of candidate lncRNAs was detected by cellular nucleoplasmic isolation,and the lncRNA TCONS₀0139850 was investigated by interference and overexpression techniques using CCK-8,EdU,flow cytometry,RNA pull down and RIP on the regulatory role of bMECs in inflammatory response.The results of the study were as follows:（1）Screening for differentially expressed genes based on RNA-seq technology:LPS can（extremely）significantly promote the expression of pro-inflammatory cytokine genes（IL-1β,IL-6 and IL-8）within bMECs.The bMECs were collected at 0 h,6 and 12 hpi of inflammation induction and subjected to RNA-seq analysis,and a total of 112 lncRNAs and 691 mRNAs were screened for differential expression.Functional enrichment analysis revealed that the above differentially expressed mRNAs regulate the body’s immune and inflammatory responses by regulating signalling pathways such as NF-κB,Toll-like receptors and TNF.（2）Expression pattern analysis of some differentially expressed lncRNAs:four differentially expressed lncRNAs（TCONS₀0039271,TCONS₀0139850,ENSBTAT00000070696 and TCONS₀0012642）were examined for their expression in the inflammatory response of bMECs.The results showed that compared to the control（0 h）,the expression of lncRNAs（TCONS₀0039271,TCONS₀0012642 and ENSBTAT00000070696）were（extremely）significantly down-regulated in LPS-induced 3,6,12 and 24 hpi bMECs;while TCONS₀0139850 was（extremely）significantly up-regulated in LPS-induced 3,6,12 and 24 hpi bMECs.（3）Sequence characterisation and subcellular localisation of candidate lncRNAs:the lncRNAs（TCONS₀0039271 and TCONS₀0139850）are involved in signalling pathways via their target genes such as mTOR,Cytokine-cytokine receptor interaction,Adherens junction and Tight junction,and are potential regulators of inflammation and immune responses.Subsequently,the sequences of the lncRNAs（TCONS₀0039271 and TCONS₀0139850）were successfully amplified.Furthermore,the lncRNAs（TCONS₀0039271 and TCONS₀0139850）were expressed in both the nucleus and cytoplasm,and mainly in the cytoplasm.（4）lncRNA TCONS₀0139850 is involved in LPS-induced inflammatory responses in bMECs:lncRNA TCONS₀0139850 promotes the expression of proinflammatory cytokines（IL-1β,IL-6 and IL-8）at mRNA and protein levels,and upregulates the expression of apoptosis marker genes（BAD,CASP3 and CASP9）,inflammation marker genes（NF-κB1 and TLR4）and chemokine genes（CXCR4 and CXCL5）at the mRNA level,and suppresses the expression of proliferation marker genes（CDK2,CDK4,PCNA,Cyclin D1 and Cyclin D2）and tight junction protein genes（TJP1,OCLN and CLDN1）at the mRNA level.These results suggest that lncRNA TCONS₀0139850 inhibits cell proliferation and vitality,alters the tight junction structure of epithelial cells,increases cell membrane permeability and exacerbates the inflammatory response.（5）A preliminary investigation of the mechanism of lncRNA TCONS₀0139850:in this study,a total of 412 interacting proteins were screened,and the interacting proteins were significantly enriched in signaling pathways such as Tight junction,PI3K-ATX and AMPK.In addition,lncRNA TCONS₀0139850 had a significant reciprocal relationship with PP2AB.In summary,this study successfully ccnstructed a model of LPS-induced inflammation in bMECs and screened 112 differentially expressed lncRNAs and 691 differentially expressed mRNAs.lncRNA TCONS₀0139850 was up-regulated in LPS-induced bMECs and exacerbated inflammation by promoting the expression of pro-inflammatory cytokines and apoptosis-related genes,etc.This study aims to provide potential therapeutic targets for the prevention and treatment of mastitis in daily cows.