Target Gene Verification Of Mir-1246 And Its Effect On The Apoptosis Of Bovine Lung Cells Under High Temperature

Mi R-1246 is a kind of micro RNA which is regulated by p53 pathway and located in the chromosome 2 long arm 31 zone 1 subband(2q31.1)of human.It involved in numerous biological process,such as development,metabolism,autoimmunization,cell proliferation and apoptosis and stress reaction.Our previous study has showed that there was differential expression of miR-1246 in heat stress and non-heat stres(P <0.05).However,the mechanism of miR-1246 regulating heat stress reaction in body is unknown.In order to reveal the effect of miR-1246 impact the regulation function of heat stress in cattle,we first utilized the bioinformatics methods to analyze the target gene of miR-1246.The results showed that c AMP response element binding protein Similarity factor(CREBL2)gene,polyprotein binding protein(PCBP2)gene,zinc finger protein factor(ZFP36L1)gene contained seed sites of miR-1246 binding in their 3'-UTR region,which suggested these genes may be miR-1246 potential target genes.Further through analysis of the potential target gene function and involved signaling pathways suggest the target genes that miR-1246 regulated are mainly enriched in cancer-related pathways.In order to verify the regulation of miR-1246 on these potential target genes,we amplified the 3'UTR sequences of three candidate target genes,compounded miR-1246 plasmid vector(miR-1246)and miR-1246 negative control vector(miR-NC).By using the liposome mediated transfect HEK293 cells and use double luciferase reporter technology,we further confirmed that CREBL2 gene and PCBP2 gene are the direct target of miR-1246,which expression was significantly down-regulated(P <0.01).The real-time PCR and Western Blot showed that miR-1246 had significant inhibitory effect on CREBL2 and PCBP2 at m RNA level and protein level(P <0.05).According to the previous study,CREBL2 gene and PCBP2 gene are related to apoptosis and growth inhibition.Finally,we utilized DAPI nuclear staining and Annexin V-FITC / PI double staining to detecte the activity of BFR-L1 cells,then found that miR-1246 could inhibit the apoptosis of bovine lung cells under heat stress.Moreover,miR-1246 could alleviate the G2/M phase arrest of heat shock bovine lung cells by cell cycle assay.In conclusion,this study revealed that miR-1246 is an important transcriptional regulator factor about body metabolic regulation under heat stress at the cellular level,Which provides a reference for finding a more effective way to resist heat stress.