| The pathogenesis and prevention and control technology of bovine mastitis are one of the hot research topics in animal husbandry.Over the years,starting from the phenotypic characteristics of mastitis,exploring reasonable management techniques and prevention and control methods has achieved certain results,but the difficult problems in the prevention and control of bovine mastitis have not been fundamentally solved.Therefore,from the molecular genetic perspective,fully elucidating the expression regulatory mechanism of key signaling molecules in the occurrence and development process of bovine mastitis,carrying out resistance breeding and developing targeted drugs are important paths for genetic improvement of cows and prevention and control of mastitis today.In the early stage of this project,a model of bovine mastitis induced by Staphylococcus aureus was established,and a key lncRNA,BMNCR(Bovine Mastitis Related lncRNA),was screened using high-throughput sequencing technology.Its significantly upregulated expression in Staphylococcus aureus-induced mastitis suggests that BMNCR plays an important role in the occurrence and development of bovine mastitis.Bioinformatics prediction revealed a binding site of bta-miR-145 on the BMNCR sequence,indicating that it may function by competitively binding bta-miR-145.At the same time,bta-miR-145 was predicted to target genes related to bovine mastitis,and ANO6 was selected as the target gene for this study.Using multiple modern molecular biology techniques such as dual-luciferase reporter assay,EdU,flow cytometry,qPCR,and Western blot,we elucidated the molecular mechanism of lncRNA BMNCR regulating bta-miR-145 at multiple levels including gene function,expression regulation,and epigenetics.We also studied the role of bta-miR-145 in regulating the occurrence and development of mastitis through the target gene ANO6,and revealed the regulatory relationship among BMNCR,bta-miR-145,and ANO6.Our study elucidated the molecular mechanisms by which the BMNCR/bta-miR-145/ANO6 pathway participates in the self-protection of bovine mastitis.The main results of this study are as follows:1、BMNCR is significantly upregulated in bovine mastitis.To identify ceRNAs that regulate mastitis,a Staphylococcus aureus infection model was established in cows,and high-throughput sequencing was performed on infected and normal mammary gland tissues to screen for differentially expressed lncRNAs.283 lncRNAs were upregulated(p<0.05),and 346 lncRNAs were downregulated(p<0.05)in mammary gland tissues with Staphylococcus aureus mastitis.The lncRNA BMNCR(Bovine Mastitis Related lncRNA)was selected for further study.The expression level of BMNCR in mammary epithelial cells after Staphylococcus aureus infection was detected using RT-qPCR,which showed that BMNCR was upregulated approximately 10-fold in mammary gland tissues infected with Staphylococcus aureus.By using RACE technology to amplify the 5’ and 3’ ends of the long non-coding RNA BMNCR.The results showed that BMNCR had a full length of 4206bp.Using biocredit analysis to locate BMNCR-related information,it was found that BMNCR was located on the third intron of TCF7L2 on bovine chromosome 26.Knocking down BMNCR in bovine mammary epithelial cells resulted in the regulation of some interleukin family inflammatory cytokines.The expression of IL-2 and IL-8 was significantly increased(p<0.01),the expression of IL-12 was significantly increased(p<0.05),the expression of IL-6 was significantly decreased(p<0.05),and the expression of IL-1α did not change significantly.The experimental results indicated that BMNCR could to some extent reduce the expression of interleukin family inflammatory cytokines and inhibit the occurrence of inflammation.The EdU cell proliferation assay verified that knocking down BMNCR significantly reduced the proliferation of mammary epithelial cells,and flow cytometry analysis showed that knocking down BMNCR promoted apoptosis of bovine mammary epithelial cells,suggesting that BMNCR plays an important role in the development of bovine mastitis.2、BMNCR targets bta-miR-145High-throughput sequencing was used to screen differentially expressed miRNAs,and the data showed that bta-miR-145 was the most downregulated miRNA in cows with mastitis induced by aureus,suggesting that bta-miR-145 is closely related to the development of mastitis.RNAhybrid was used to predict the potential binding sites between BMNCR and bta-miR-145,and the targeting relationship between them was further verified by a dual-luciferase reporter assay.Knocking down BMNCR significantly increased the expression of bta-miR-145.To verify the function of bta-miR-145,bta-miR-145 inhibitor was transfected,and the expression levels of IL-1α,IL-2,and IL-12 in bovine mammary epithelial cells were significantly reduced(p<0.05),indicating that bta-miR-145 inhibits the occurrence of inflammation to a certain extent.Knocking down bta-miR-145 significantly increased the proliferation of bovine mammary epithelial cells,while transfecting bta-miR-145 mimic inhibited cell proliferation and promoted apoptosis,indicating that it is involved in regulating various biological functions,including cell proliferation and apoptosis.3、bta-miR-145 targets ANO6Predict target genes for bta-miR-145 by using microRNA target gene prediction software tools.As the focus of this study was on immune function or inflammation,64 genes were evaluated for their function and pathways through GeneCards and NCBI websites.Finally,one gene related to immunity or inflammation,ANO6,was selected for further study.The binding site between miR-145 and ANO6 was verified by dual luciferase reporter assay.qPCR,Edu,and flow cytometry were used to validate that ANO6 has a certain inhibitory effect on inflammation,promotes the proliferation of bovine mammary epithelial cells,and inhibits cell apoptosis,suggesting that ANO6 is closely related to the occurrence of mastitis.In summary,BMNCR can regulate the expression of ANO6 through the sponge effect on bta-miR-145,thereby affecting the proliferation and apoptosis of bovine mammary epithelial cells.Based on the ceRNA theory,we constructed a new regulatory network of BMNCR/bta-miR-145/ANO6 in bovine mammary epithelial cells.When cows are infected with Staphylococcus aureus-induced mastitis,the host initiates a self-protective mechanism and the mammary tissue produces inflammation.At this time,BMNCR is significantly upregulated,inhibiting the expression of bta-miR-145 and increasing the expression level of ANO6,while affecting the expression of inflammatory factors.This leads to a significant increase in the proliferation of bovine mammary epithelial cells and a reduction in apoptosis,promoting the recovery of damaged tissue after bacterial invasion.This research can provide benefits for the precise molecular breeding of cows and the development of targeted drugs for mastitis,and has important implications for increasing milk production and improving milk quality. |
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