## 2 Results
### Direct Interaction between the Predicted miRNAs and the 3'-UTR of PCSK7
This could be due to several factors such as the accessibility of different miRNA target sites in the cells, i.e., when one miRNA targets the mRNA, the site for another miRNA might not be accessible. Therefore, we omitted the mix microRNA analyses for further analysis. Direct Interactions between miR-125a-5p, miR-143-3p, and miR-409-3p with the 3\({}^{\prime}\)-UTR of PCSK7  
A luciferase reporter assay further confirmed that miR-125a-5p, miR-143-3p, and miR-409-3p directly targeted the 3\({}^{\prime}\)-UTR of the _PCSK7_ transcript since the negative regulatory effect was lost upon the deletion of the miRNA target sites (miR-125a-5p, miR-143-3p, and miR-409-3p) on the 3\({}^{\prime}\)-UTR of the _PCSK7_ reporter plasmid (Figure 5). In this regard, three different mutated miR plasmids were constructed for both miR-125a-5p and miR-143-3p. Single-target sites of each miRNA were omitted individually in two separate plasmids, and the two putative binding sites of the miRNAs were deleted in the other plasmid. The data demonstrate that overexpression of miR-125a-5p still decreased the relative luciferase activity in the HEK293T cells co-transfected with the plasmids carrying a mutated form of _PCSK7_ 3\({}^{\prime}\)-UTR, containing a single deletion of miR-binding sites (Mut-a and Mut-b), compared with cells co-transfected with the mock counterpart vectors along with the same mutant vectors (Mut-a and Mut-b) (\(p\) = 0.04 and \(p\) = 0.01). In contrast, a mutated form of _PCSK7_ 3\({}^{\prime}\)-UTR, featuring the deletion of both miRNA target sites (Mut-a,b), failed to significantly decrease the relative luciferase activity in these cells (\(p\) = 0.24) (Figure 5A). As was observed for miRNA-125a-5p, the overexpression of miR-143-3p had no significant effect on luciferase activity when the two putative target sites (Mut-a,b) were omitted from the 3\({}^{\prime}\)-UTR of _PCSK7_ in HEK293T cells (\(p\) = 0.98). By comparison, luciferase activity was  
Figure 4: Luciferase assay results following co-transfection of the miR-overexpressing vector and the 3\({}^{\prime}\)-UTR of the wild-type _PCSK7_ in HEK293T cells. Relative luciferase activity considerably decreased after the overexpression of mir-125a-5p, miR-143-3p, and miR-409-3p; however, there was no significant alteration in relative luciferase activity for miR-320a-3p and miR-224-5p, which were used as controls. \({}^{\star}\), \({}^{\star\star}\), and \({}^{\star\star\star}\) stand for \(p\) value less than 0.05, 0.01 and 0.0001.  
considerably diminished in these cells expressing the miR-143-3p plasmid and mutated vectors carrying only one miRNA response element sequence (Mut-a; \(p\) = 0.03 and Mut-b; \(p\) = 0.004) (Figure 5B). Finally, miR-409-3p overexpression did not lead to a reduction in luciferase activity when the only putative target site of miR-409-3p was absent from the 3\({}^{\prime}\)-UTR of _PCSK7_ (\(p\) = 0.40) (Figure 5C). Overall, the above results confirm the presence of two independent miRNA response elements for both miR-125a-5p and miR-143-3p and one miRNA response element for miR-409-3p as active elements. We conclude that miR-125a-5p, miR-143-3p, and miR-409-3p could regulate the expression of _PCSK7_ mRNA through direct interactions with these target miRNA-binding sites.