| Mastitis in dairy cattle is mainly caused by the invasion of pathogenic bacteria into the mammary gland,which has a high incidence,expensive treatment and low cure rate,and seriously affects milk production and quality,bringing great losses to the modern dairy industry and farm economic development.Long non-coding RNAs(lncRNAs)are a newly discovered class of non-coding RNAs that have been shown to be involved in the regulation of various biological processes at the transcriptional or post-transcriptional level and are closely associated with mastitis in dairy cows.However,only a few differentially expressed lncRNAs have been functionally validated regarding mastitis in dairy cows so far.In this study,intracellular lncRNA CA12-AS1 was overexpressed and knocked down in bovine mammary epithelial cells(bMECs),and qPCR,ELISA,CCK-8,EdU and flow cytometry were used to investigate the role of lncRNA CA12-AS1 in the regulatory role of LPS-induced inflammatory response in mammary epithelial cells of dairy cows.The main findings are as follows:(1)Amplification primers for lncRNA CA12-AS1 sequence were designed and cloning sequencing of lncRNA CA12-AS1 was performed,and the results showed that a lncRNA CA12-AS1 fragment of 1 131 bp was successfully amplified,indicating that the lncRNA is real and mainly localized in the cytoplasm.KEGG annotation analysis of the target gene of lncRNA CA12-AS1 indicated that lncRNA CA12-AS1 may be involved in breast cancer,bacterial invasion of epithelial cells,Hippo,Tight junction(TJ)and Notch pathways.(2)The expression level of lncRNA CA12-AS1 in the inflammatory response induced by LPS in bMECs was detected by qPCR.The results showed that the expression of lncRNA CA12-AS1 was highly significantly upregulated at 3,6 and 12 h(P<0.01)and at 24 h(P<0.05)compared with the control group,suggesting that lncRNA CA12-AS1 is involved in regulating the inflammatory response of bMECs.(3)The overexpression and inhibition of lncRNA CA12-AS1 in bMECs were performed by constructing lncRNA CA12-AS1 overexpression vector and synthesizing specific siRNA in vitro,while LPS was used to induce inflammatory responses in bMECs,and the expression of inflammatory factors and TJ signaling pathway-related genes,inflammatory factor secretion,cell viability,proliferative capacity and apoptosis were detected.The results showed that overexpression of lncRNA CA1-AS1 significantly promoted the expression of inflammatory factors at the mRNA and protein levels,apoptosis-related genes(BAX,caspase3 and caspase9)and proliferation-related genes(CDK2,CDK4 and PCNA)mRNA,and inhibited the expression of TJ signaling pathway-related genes(Claudin-1,Occludin and ZO-1),and mRNA expression levels of the apoptotic gene BCL2;meanwhile,the secretion of cellular inflammatory factors and apoptosis were promoted,and the viability and proliferative capacity of bMECs were inhibited.And inhibition of lncRNA CA12-AS1 obtained the opposite result to overexpression of lncRNA CA12-AS1.(4)A dual luciferase reporter gene assay was used to identify lncRNA CA12-AS1 downstream target miRNAs,and the results showed that lncRNA CA12-AS1 binds to miR-133a target.In the LPS-induced inflammatory response of bMECs,overexpression of miR-133a suppressed the expression of inflammatory factors(IL-6,IL-8 and IL-1β),pro-apoptotic genes BAX and TJ signaling pathway-related genes(Claudin-1,Occludin and ZO-1)at the mRNA level,promoting cell viability and inhibiting apoptosis.In summary,lncRNA CA12-AS1 is a pro-inflammatory gene in bMECs,and the results of the study promote LPS-induced inflammatory response in bMECs by targeting miR-133a elucidated the function of lncRNA CA12-AS1 in dairy mastitis at the cellular level,providing ideas for the molecular treatment of dairy mastitis and laying a foundation for breeding for mastitis resistance in dairy cows. |
PDF Full Text Request