Identification Of Target Genes Of MiR-214 And It’s Immune Regulation On Bovine Mammary Epithelial Cells

MicroRNA is a class of non-coding small RNA with regulation function, and the length is 20~25 base. MicroRNA connects with target genes 3 ’UTR by complementary base pairing,deterring target mRNA degradation or inhibiting protein translation, so as to regulate the expression of target genes. We transfected miR-214 mimics and plasmid of target genes about immunity to 293 A cell line, detecting dual-luciferase activity after 48 hours to screen miR-214 potential target genes. Then we transfected miR-214 mimics or miR-214 inhibitor to bovine mammary epithelial cells, extracting total RNA and reversing transcription after 24 hours. We detected the expression of target genes, its downstream pathway genes and inflammatory cytokines by quantitative real-time PCR. Main results were as follows:In the experiment of transfecting 293 A cell line, the dual-luciferase activity of the experimental group of NFATc3, TRAF3 significantly lower than the control group(p<0.01),yet the experimental group and the control group of TOLLIP, IGDCC3, NFATc4, IL17 RD have no significant difference. We speculated that NFATc3, TRAF3 are the targets of miR-214.In the experiment of transfecting miR-214 mimics to bovine mammary epithelial cells,the expression of the experimental group of target gene NFATc3, TRAF3, downstream pathway gene MAP3K14, TBK1 and inflammatory cytokines IL-6, IL-1β significantly lower than the control group(p<0.01). For transfecting miR-214 inhibitor to bovine mammary epithelial cells, the expression of the experimental group of target gene NFATc3, TRAF3,downstream pathway gene MAP3K14, TBK1 and inflammatory cytokines IL-6, IL-1βsignificantly higher than the control group(p<0.05). These results proved that in bovine mammary epithelial cells, miR-214 signally inhibits the expression of target gene NFATc3,TRAF3 and indirectly inhibits the expression of downstream pathway gene MAP3K14, TBK1 and inflammatory cytokines IL-6, IL-1β.In conclusion, we expounded the function of miR-214 and its molecular regulation mechanism in cellular level. The results accumulated data for molecular regulation network of dairy cow mastitis, and provided new molecular basis and scientific guidance for molecular breeding of mastitis resistance.