To further test the hypothesis that miR-9 downregulates BK mRNA by binding directly to the BK mRNA 3’UTR-2.1 region, we constructed BK plasmids containing the rat BK 3’UTR region with or without miR-9 MRE (miR-9 MRE+, miR-9 MRE−, respectively; based on the AF135265 sequence) and transfected HEK293 (which lack endogenous miR-9 and BK) with each of these constructs in the presence or absence of miR-9 and miR-135b (a miRNA with poor complementarity to BK mRNA, Figure 5G). This approach revealed that miR-9 MRE in the BK-3’UTR is required for miR-9 specific downregulation of BK mRNA, as determined by qRT-PCR (Figure 5H).
