克柔念珠菌诱导奶牛乳腺上皮细胞lncRNA差异表达分析OA

[Objective]Long non-coding RNAs(lncRNAs)have important regulatory roles in host intrinsic immune responses,but their expression patterns in Candida krusei(C.krusei)infected mammary alveolar cells-large T(MAC-T)cell lines of dairy cows have not been clarified.The aim of this study was to characterize the features and functions of differentially expressed lncRNA(DElncRNA)in C.krusei infected MAC-T,screen for potential biomarkers and explore the immune regulatory pathways involved.[Method]MAC-T were divided into blank control group(cultured conventionally in DMEM medium only)and C.krusei ATCC 6258 infection group(co-cultured with C.krusei ATCC 6258 at a multiplicity of infection(MOI)=1).Both groups comprised three biological replicates.Transcriptome sequencing(RNA-Seq)was employed to analyse the lncRNA expression profiles of both cell groups.DElncRNAs were identified using bioinformatics methods,with target genes predicted via TargetScan and miRanda software.This was followed by GO functional and KEGG pathway enrichment analysis.Concurrently,ELISA measured interleukin-1 beta(IL-1β),IL-18,tumor necrosis factor-α(TNF-α),IL-4,and IL-6 expression in cell supernatants to validate the inflammatory model establishment.Ten DElncRNAs were selected for Real-time quantitative PCR validation to confirm sequencing reliability.[Results]Sequencing identified a total of 3 425 lncRNAs,among which 55 significantly upregulated and 79 significantly downregulated DElncRNAs were screened in infected group compared to blank control group.GO functional enrichment analysis indicated that quercetin exerted its effects through biological processes such as positive regulation of inflammatory response and positive regulation of IL-6 production in locations including the extracellular space and extracellular region.KEGG pathway enrichment analysis predicted that potential target genes of DElncRNAs might be involved in 67 signalling pathways,with significant enrichment observed in inflammation-related pathways such as PI3K-Akt,NF-κB,and MAPK,as well as the ferroptosis pathway.Real-time quantitative PCR results aligned with gene expression trends observed in RNA-Seq data.Notably,the significantly upregulated lncRNA MSTRG.11194 modulates cellular inflammatory responses and ferroptosis by targeting WDR74 and SLC3A2 genes.[Conclusion]In this study,134 DElncRNAs associated with C.krusei infection were identified in the mammary epithelial cells of dairy cows,which mightbe involved in the inflammatory response and ferroptosis of the mammary epithelial cells of dairy cows through the targeted regulation of immune signalling pathways.This study provided theoretical basis for the elucidation of the mechanism of lncRNAs regulating the C.krusei induced mastitis in dairy cows,and the development of a new type of molecular markers.