Abstract
miRNAs are a class of endogenous small non-coding RNAs, approximately 22 nucleotides in length, which play a role in post transcriptional regulation by target mRNA degradation or inhibiting their translation. glycerol-3-phosphate acyltransferase 1, mitochondrial (GPAM) that catalyze the initial and committed step in glycerolipid biosynthesis has long length of 3´untranslated region (3´UTR) could be found a number of SNPs and affected by miRNA. Therefore, in this study, we aimed to investigate whether bta-miR-2468 binds to the 3´UTR of GPAM, including the g. 55930 C>T SNP, and whether each allele of this SNP affects the binding affinity between the target miRNA and the 3´UTR. For the dual luciferase assay, pmirGLO-55930 C and pmirGLO-55930 T as plasmid DNA includes g. 55930 C>T react bta-miR-2468, identified protein expression with firefly luciferase activity and renilla luciferase activity. As a results, validate the results reported by in silico assay that reported when either g. 55930 C or g. 55930 T were present in the seed region of bta-miR-2468, there was an evident reduction, on the contrary, co-transfection of both the g.55930 SNP allele and bta-miR-2468 did not result in significantly different luciferase activities in HEK293T cell. In this study, we demonstrated the importance of post-transcriptional regulation of GPAM gene expression by bta-miR-2468, Considering the effect of haplotype (g. 54853 A>G - g. 55441 A>G - g. 55930 C>T), the effect of the g.55930 T>C SNP should be evaluated in future studies using pmirGLO vectors, including haplotypes.
Figures & Tables
Fig. 1. bta-miR-2468 binding to GPAM 3´UTR. (a) Schematic representation of the dual luciferase assay for C and T alleles. (b) Relative luciferase activities are similar to C and T alleles with bta-miR-2468, as compared with negative control miRNA in HEK293T cells. Error bars (SE) are derived from three independent experiments performed in triplicate, and data were compared using a Student’s t-test. * <0.05
