Expression Of Mastitis Related MiR-223 In Dairy Cow And Its Roles In Inflammation Of Bovine Mammary Epithelial Cells

Cow mastitis is an inflammatory disease caused by pathogenic microorganisms in mammary tissue.Its incidence rate is high,its cure rate is low,and its recurrence is easy.It will cause milk production to drop and milk quality to decrease,which will cause great economic losses to the modern dairy industry.In recent years,domestic and overseas researchers have found that the occurrence,development process,susceptibility and disease resistance of mastitis are regulated by a gene network composed of multiple genes,among which the NF-κB signaling pathway is an important regulatory pathway.However,the molecular regulation mechanism of these genes or signaling pathways on dairy cow mastitis is not fully understood,consequently hindhindering efficiency of molecular breeding for mastitis resistance in dathcowsry cow.In this study,the experimental inflammatory cell model of bovine mammary epithelial cells(bMECs)induced by LPS was successfully constructed.On this basis,the changes in miR-223 expression quantity under LPS stimulation were detected.In addition,many methods,including Gene overexpression or silencing,qPCR,ELISA,Western Blotting,CCK-8,and flow cytometry and function were used to explore the function of miR-223 in the LPS-stimulated bMECs inflammation model.The results were as follows:(1)The potential target genes of miR-223 were analyzed by Targets can and miRwalk software,and the signal pathways enriched by these potential target genes were analyzed by the Cluster Profiler software package(R-3.2.4).The results showed that the potential target gene of miR-223 may be involved in regulating cell proliferation,differentiation,and inflammatory response through the NF-κB inflammatory signaling pathway.(2)The CHECK-2 double luciferase reporter gene vectors of 7 mRNA(RHOB,PARP1,ACVR2A,ZNF395,ATP2B1,BRPF3 and NLRP3)-3’UTR were successfully constructed,and the target gene was identified by double luciferase reporter gene experiment.The results showed that RHOB was the target gene of miR-223 in dairy cows.(3)bMECs were induced by LPS,and the expression quantity of related inflammatory genes was detected by qPCR.The results showed that the mRNA levels of inflammatory cytokines IL-1β,IL-6 and IL-8 were significantly up-regulated(P<0.01),indicating that LPS initiated the inflammatory response of bMECs,and successfully constructed the inflammation model of bMECs.(4)In the LPS-induced bMECs inflammation model,the expression quantity of miR-223 was significantly up-regulated(P<0.01)the in inflammatory cell model,and targeted inhibited the expression of RHOB gene mRNA and protein(P<0.01).Overexpression of miR-223 in inflammatory bMECs can significantly inhibit the expression level of inflammatory signaling pathway TLR4 at the mRNA level(P<0.01),and the expression level of inflammatory cytokines(IL-1β,IL-6,IL-8)and the inflammatory signaling pathway NF-κB were significantly inhibited at the mRNA and protein levels(P<0.01),while the opposite results were obtained by inhibiting miR-223.In addition,overexpression of miR-223 in inflammatory bMECs significantly inhibited the expression of the proapoptosis-related gene BAX(P<0.01)and promoted the expression of the inhibited apoptosis-related gene BCL2(P<0.05),while the promotion of BCL2 by silencing miR-223 did not show the opposite results.Therefore,it was further verified by flow cytometry,and the results showed that overexpression of miR-223 could inhibit the apoptosis of bMECs(P<0.01)and promote the proliferation of bMECs(P<0.05).(5)The miR-223 target gene RHOB gene was interfered with in the LPS-induced bMECs inflammation model,besides the expression of inflammatory cytokines and inflammatory signaling pathway genes was detected by qPCR technology,and the secretion of inflammatory factors was detected by ELISA technology.The apoptosis of bMECs was detected by flow cytometry,and it was found that the detection results of the above indicators after interfering with RHOB were completely consistent with the results of overexpression of miR-223,indicating that miR-223 indeed regulates the inflammatory response and apoptosis of bMECs by inhibiting the expression of the target gene RHOB.In conclusion,miR-223 is an anti-inflammatory gene of bMECs,which can alleviate the inflammatory response of bMECs by targeting the expression of RHOB and NF-κB,and miR-223 also promoted the proliferation of bMECs and inhibited the apoptosis of bMECs by targeting RHOB.The study clarifies the function of miR-223 in dairy cow mastitis at the cellular level,which can provide new ideas for molecular therapy and disease-resistant molecular breeding of dairy cow mastitis,and has potential application value.