Expression And Role Of LncRNA In Inflammatory Response Of Bovine Mammary Epithelial Cells

Bovine mastitisis a complex inflammatory disease,which seriously harms the dairy industry and the development of dairy industry.In order to completely reduce the incidence of bovine mastitis,researchers have carried out a series of studies on its clinical symptoms,pathogenic factors and diagnosis and treatment.The pathological process of bovine mastitisis affected by nutrition,heredity and environment,but its pathological mechanism has not been fully understood.Long noncoding RNA（IncRNA）is a genetic regulatory molecule discovered in recent years that can regulate immunity and is involved in the regulation of a variety of inflammatory diseases in humans and model animals.However,its regulatory effect on the pathological process of bovine mastitis remains unclear.The bovine mammary epithelial cell line（MAC-T）was used as the research object,and lipopolysaccharide（LPS）induction was used to establish the model of bovine mastitis.To investigate the expression and effect of candidate lncRNAs on MAC-T cells in inflammatory response.The unknown candidate lncRNAs were screened layer by layer through expression profile analysis,bioinformatics prediction,RNA interference and other techniques,and 1 key lncRNA was selected for functional verification.The following research results were obtained:（1）Inflammatory cell model was successfully established,and inflammatory cytokines IL-6,IL-8 and TNF-α were significantly up-regulated in inflammatory MAC-T cells.Twelve candidate IncRNAs were differentially expressed in clinical mastitis tissues and inflammatory MAC-T cells of dairy cows by qPCR.Significantly differentially expressed lncRNAs（TCONS₀0100120,TCONS₀0019437,TCONS₀0009489 and TCONS₀0066712）were selected for subsequent studies.（2）IncRNA target genes and GO analysis indicated that potential target genes of TCONS₀0100120,TCONS₀0066712 and TCONS₀0019437 were involved in biological processes related to breast development,inflammatory response and immune regulation.Interaction network analysis indicated that TCONS₀0100120 may regulate cell proliferation gene CDK2 and inflammation gene TLR4.Since TCONS₀0100120 is the reverse complement of C3Hlorf162-AS1gene,and TCONS₀0100120 is the antisense IncRNA of this gene,it is named IncRNA C3Hlorf162-AS1.KEGG annotation suggests that C3H1orf162-AS1 may be involved in cancer,Rapl,PI3K/AKT,AMPK and regulating stem cell multifunctional pathways.（3）lncRNA C3H1 orf 162-AS1 was highly up-regulated in both clinical mastitis tissues and inflammatory MAC-T cells of dairy cows.The detection of interference efficiency of three interference sequences showed that si-1 had the best effect and the interference efficiency reached 70%,which was used in subsequent experiments.Compared with si-NC group,interfering C3H1 orf 162-AS 1 could decrease the expression of proliferative genes CDK2 and CDKN1A,decrease the expression of anti-apoptotic gene BCL-2,increase the expression of pro-apoptotic gene BAD,and promote the expression of inflammatory genes NFKB1,TLR4,AKT1,GNAI and NF-κB.It decreased the expression of AHUK,and promoted the expression of inflammatory cytokines IL-6,IL-8 and TNF-α.In conclusion,the key lncRNA C3H1 orf 162-AS 1 regulating inflammation in bovine mammary epithelial cells was successfully screened and identified from 12 unknown lncRNAs in this study,and the possible mechanism of its regulation of bovine mastitis was explored to provide a reference for revealing the complex gene regulation network of bovine mastitis.