Expressin Of MiRNAs In The LPS-induced Inflammation In Mammary Epithelial Cell And Preliminary Study On MiR-223 Act As A Modulator In Toll-like Signaling Pathway

microRNA(miRNA)is a kind of non-protein-coding,single stranded smallRNA which has approximately 20~25 nt in size.It is highly conserved,specific temporal and spatial expression,and its expression is strictly regulated.MiRNAs proceed to complementary pairing,inhibiting target mRNA transcription,translation or inducing its degradation by binding directly to the 3'untranslated region(3'untranslated regions,UTRs)of the target mRNA molecule,so that in posttranscriptionally regulate gene expression level.Studies have shown that miRNAs involved in the regulation of many physiological disease processes,and its expression disorder is closely related to the mechanism of disease.Ruminant mastitis,particularly bovine mastitis,has not been effectively resolved worldwide,which seriously affectes the development of animal husbandry and dairy industry.Studies have shown that the expression of miRNAs has significantly changed when udder inflammation occured,which reveals miRNA may be involved in the regulation of mammary gland inflammation,but the exact mechanism has not been clear.The aim of this study is to determine whether a number of inflammation-related miRNAs have been involved in LPS-induced inflammation of the mammary gland,and make a preliminary study on the role of miRNA-223 in the Toll-like receptor signaling pathways.The results of this study are expected to provide experimental basis for new targets discovery which can prevent and treat ruminants mastitis.In this study,difference concentrations and doses of LPS stimulate mouse mammary epithelial cells(Ep H4-Ev)48h,qRT-PCR detection of inflammation-related miRNAs(miR-223,miR-146 a,miR-181 and miR-155)expression changes.qRT-PCR test results show that,LPS-induced Ep H4-Ev cell inflammatory response,the four kinds of expression of inflammation-related miRNAs were significantly increased(P<0.05),and showed varying degrees of dose-dependent.Pre-miR-223 gene was cloned in mouse genomic DNA of mammary epithelial cells byRT-PCR;Construction of the plasmid Minicircle,Piggy Bac transposons and pc DNA3.1(+)for the female three miR-223 eukaryotic expression vector,and transfected into Ep H4-Ev cells,by observing the fluorescence of GFP,qRT-PCR detection miR-223 gene mRNA expression levels in Ep H4-Ev cells,the results revealed the successful construction Mini-miR-223,PB-miR-223 and pc D-miR-223 three eukaryotic expression vector.To explore the influence of miR-223 on TLR signaling pathway,we transfected miR-223 overexpression vector and miR-223 inhibitor into mouse mammary epithelial cells(Ep H4-Ev).The expression changes of molecules involved in TLR signaling pathway have been determined with qRT-PCR,and Western blot has been used to detect changes in their protein levels.The experimental results showed that after the recombinant plasmid of miR-223 transfected,compared with its PB no-load group,the expression of TLR4,NF-?B,IL-6 reduced;the expression of My D88,TNF-? significantly decreased(P<0.05),expression of IL-1? was significantly reduced(P<0.001).miR-223 inhibitor did the opposite acts compared with the overexpression of miR-223.The results of western blot showed that overexpression of miR-223 in the PB group,the content of IL-6 significantly decreased(P<0.05),compared with the control group;overexpression of miR-223 in the pc DNA3.1(+)group,content of IL-6 showed a downward trend.Content of NF-?B in both PB and pc DNA3.1 did not significantly change,compared to control group.In conclusion,miR-223 may play an anti-inflammatory role in TLR signaling pathway through inhibiting the expression of IL-6,but whether IL-6 is the target genes of miR-223 requires experimental verification further.