Identification of the interaction between bta-miR-370 and OLR1 gene in bovine adipocyte.

ABSTRACT

It has been shown that the oxidized low density lipoprotein receptor 1 (OLR1) gene plays an important role in the degradation of oxidized low density lipoprotein. Previous studies found a SNP in the 3'-untranslated region (3'-UTR) of the OLR1 gene associated with milk production traits in different dairy cattle populations and with loin eye area and marbling depth in beef cattle. MicroRNAs can regulate gene expression by binding the 3'-UTR of target genes to degrade or to repress the translation of target genes. Bioinformatics have shown that there is a binding site of bta-miR-370 in the 3'-UTR of the OLR1 gene, and a previous luciferase reporter assay system showed that the A/C mutation occurring in the 3'-UTR of this gene caused the binding sites of bta-miR-370 to disappear in HEK293 cells. To further validate whether OLR1 was the target gene of bta-miR-370, the over-expression and interference expression of bta-miR-370 were determined by transfecting bta-miR-370 mimics and inhibitor supplementations into bovine adipocyte. The qRT-PCR result showed that the relative expression of OLR1 gene significantly decreased in the mimics group compared to the control, whereas the expression level in inhibitor group was higher than its control group. The above results were further verified by a Western blot at the protein level. In addition, lipid formation analysis of bovine adipocytes was performed via oil red O staining, and we found that cytoplasm lipid droplets in the inhibitor group showed a tendency to increase compared to the control group, whereas in the mimics group, we observed an obvious decrease of cytoplasm lipid droplets compared to the control and inhibitor groups. Taken together, our data here suggest that bta-miR-370 has a negative regulation role for OLR1 both at the gene expression and protein levels and bovine adipocytes cytoplasm lipid droplets formation, which provides a reference for illustrating how the OLR1 gene affects milk production and beef quality traits in cattle.