Experimental identification of microRNA targets on the 3′ untranslated region of human FMR1 gene

FMR1 gene plays an important role in the development of central nervous system. Down-regulation of the FMR1 expression leads to fragile X syndrome. MicroRNAs (miRNAs) can repress gene expression by base pairing with their mRNA targets. By computer programs analysis, five miRNAs: miR-19a, miR-19b, miR-142, miR-302b* and miR-323-3p potentially target to different sites on the FMR1 3′ untranslated region (3′ UTR), except that miR-19a and miR-19b share the same targeting site. To test whether these miRNAs repress reporter gene expression by interacting with the miRNA targets on the FMR1 3′ UTR, we developed two chimeric constructs: one construct expressing a firefly luciferase with the FMR1 3′ UTR or its miRNA target mutations and the other construct expressing a pre-miRNA fusing with GFP. Luciferase assay co-transfecting with the two constructs showed that the miRNAs, miR-19b, miR-302b* and miR-323-3p could repress gene expression in HEK-293 cells, suggesting a role of these miRNAs in the regulation of the FMR1 expression. The constructs used in this study can be widely used to identify the miRNA targets in any interested genes, which will greatly promote the current progress in understanding the biological function of miRNAs.