Functional Validation Of Mastitis Resistance-associated MiR-2448-3p And Its Target Gene ADIPOQ In Xinjiang Brown Cattle

Mastitis in cattle is an inflammatory response that is largely regulated by epigenetic factors,of which non-coding RNA has been the focus of much research in recent years.Currently,a large number of studies have shown that miRNAs play an important regulatory role in the immune system,but they have been less studied in bovine mastitis,especially in Xinjiang brown cattle(XJBC).Therefore,based on the transcriptome sequencing results of the group,we screened miRNAs associated with mastitis resistance in XJBC and constructed a bovine mammary epithelial cell inflammation model using Lipopolysaccharide(LPS),a component of Gram-negative bacteria,and Lipoteichoicacid(LTA),a component of Gram-positive bacteria.The biological functions of key candidate miRNAs and their target genes were verified by real-time fluorescence quantification,overexpression,inhibitor,dual-luciferase reporter gene,CCK-8 and other experiments to provide some theoretical basis for the application of non-coding RNAs in molecular breeding related to bovine mastitis resistance.The following are the main results obtained from this study,(1)12 miRNAs were randomly selected based on the sequencing results of miRNAs in the RNA-seq results,and the sequencing results were verified by quantitative assays.The results showed that the sequencing results were all consistent with the quantitative results,which proved that the sequencing results were true and reliable and could be used in this experiment.After screening,miR-2448-3p was finally obtained for the subsequent validation assay.(2)After stimulation of bovine mammary epithelial cells with 10 ng/μL of LPS and 25 ng/μL of LTA for 3 and 9 h,the results showed that the expression level of miR-2448-3p reached the highest level and the expression of related cellular inflammatory factors(IL-1β、IL-6、TNF-α)was significantly up-regulated,a model of cellular inflammation was successfully constructed.(3)Over-expression of miR-2448-3p promoted the development of LPS-and LTA-induced inflammation in bovine mammary epithelial cells;reduced miR-2448-3p expression inhibited the development of LPS-and LTA-induced inflammation in bovine mammary epithelial cells.(4)The target gene prediction results showed that the target genes of miR-2448-3p were significantly enriched in signalling pathways associated with inflammatory responses.The dual luciferase reporter gene assay demonstrated a targeting relationship between miR-2448-3p and the ADIPOQ gene,and a 67-bp copy number variation(CNV)in the promoter region of this gene was significantly correlated with milk production traits in XJBC.In summary,miR-2448-3p is involved in the inflammatory response of bovine mammary epithelial cells.Inhibition of miR-2448-3p can reduce the expression of cellular inflammatory factors.In addition,miR-2448-3p can target binding to the ADIPOQ gene,and the variant sites in the promoter region of this gene can be used as molecular markers for the improvement of milk production traits and disease resistance traits in XJBC.This study provides a theoretical basis for further research on the molecular mechanisms of miRNAs in mastitis disease resistance traits in XJBC.