The Function of lncRNA RRAS2-AS1 in LPS Induced Bovine Mammary Epithelial Cells Inflammation
[Background]Bovine mastitis is one of the most serious diseases in dairy cows,which seriously affects milk quality and not only causes economic losses,but also even jeopardizes human health.Previous studies have shown that lncRNAs were widely involved in inflammation and immune regulation in humans and animals.lncRNA RRAS2-AS1 was a newly identified differentially expressed lncRNA in our previous study,and its expression pattern and function are still unclear.[Objective]The aim of this study was intended to investigate the mechanism of lncRNA RRAS2-AS1 in the inflammatory response of bovine mammary epithelial cells(bMECs)in dairy cows,so as to provide a theoretical basis for the resolution of molecular regulatory mechanisms of bovine mastitis and molecular breeding for anti-mastitis.[Method]The cloning of lncRNA RRAS2-AS1 was performed by RT-PCR and RACE,and the target genes prediction and functional enrichment analysis of lncRNA RRAS2-AS1 were performed by bioinformatics methods.The bMECs were identified by immunofluorescence staining,and the subcellular localization of lncRNA RRAS2-AS1 was detected by cytoplasmic isolation and semi-quantitative PCR.The expression patterns of lncRNA RRAS2-AS1 in lipopolysaccharide(LPS)induced bMECs inflammatory response and in bovine mammary tissues with mastitis were detected using qRT-PCR.An inflammatory cell model was constructed using LPS-induced bMECs,on this basis,the effects of lncRNA RRAS2-AS1 on the expression of pro-inflammatory cytokine genes,proliferation-related genes and apoptosis-related genes at the mRNA and/or protein levels in inflammatory bMECs were studied using lncRNA overexpression,qRT-PCR and ELISA techniques.At the same time,the effects of lncRNA RRAS2-AS1 on the proliferation,viability,and apoptosis of bMECs were further verified using EdU,CCK-8 and flow cytometry.[Result]The length of lncRNA RRAS2-AS1 was 363 bp,which was mainly localized in the cytoplasm.The expression assay results showed that,compared with the control group,the expression of lncRNA RRAS2-AS1 was significantly down-regulated in LPS induced bMECs inflammation and in bovine mammary tissues with mastitis(P＜0.05).The results of lncRNA RRAS2-AS1 overexpression assay showed that,compared with the control group,the expression of key genes the inflammatory signaling pathway(TLR4 and NF-κB1),pro-inflammatory cytokine genes(IL-1β,IL-6 and IL-8),pro-apoptotic genes(BAD,CASP3,BAX,etc.)were significantly down-regulated(P＜0.01)in the lncRNA RRAS2-AS1 overexpression group,while the expression levels of proliferation marker genes(CDK2,CDK4,and PCNA)were significantly up-regulated(P＜0.05).In addition,the cell viability of the lncRNA RRAS2-AS1 overexpression group was significantly increased(P＜0.05),while the apoptosis rate of bMECs was significantly reduced(P＜0.01).[Conclusion]lncRNA RRAS2-AS1 was significantly down-regulated in LPS induced bMECs inflammation and in bovine mammary tissues with mastitis.Overexpression of lncRNA RRAS2-AS1 decreased the expression of pro-inflammatory cytokines IL-6,IL-8,and IL-1β at mRNA and protein levels,and promoted cell viability and proliferation and inhibited apoptosis,which attenuated the inflammatory response of LPS-induced bMECs.These and the results laid the foundation for analyzing the molecular regulatory network of mastitis in dairy cows.
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