## 2 Results
### Functional Effects of miR-125a-5p on PC7 Activity  
Western blot analysis was conducted to determine whether miR-125a-5p affected the functional activity of endogenous PC7 in Huh7 cells. A wild-type human PC7-overexpressing vector was co-transfected with pre-miR-125a-5p and pre-miR-224-5p-overexpressing plasmids in Huh7 cells. Western blot results at 48 h post-transfection revealed that overexpressed PC7 protein levels fell significantly by \(\sim\)80% (\(p\) = 0.0025) following miR-125a-5p co-expression, compared with the mock-related (pEGFP  
Figure 5: Luciferase assay analysis of cells co-transfected with the miR-overexpressing vector and miRNA target-site-deleted (Mut) psiCHECK-2 vectors in the HEK293T cells. The results demonstrate a direct interaction of miR-125a-5p, miRNA-143-3p, and miR-409-3p with the 3\({}^{\prime}\)-UTR of _PCSK7_. (**A**) Both target sites of miR-125a-5p in the 3\({}^{\prime}\)-UTR were functional. There was no significant alteration in relative luciferase activity in cells transfected with miR-125a-5p, Mut-a,b in the 3\({}^{\prime}\)-UTR of the psiCHECK-2 vector, in which both target sites in miR-125a-5p were omitted. Relative luciferase activity significantly decreased following miR-125a-5p overexpression in Mut-a,b in the 3\({}^{\prime}\)-UTR of the psiCHECK-2 vector, in which only one target site of miR-125a-5p was deleted. (**B**) The two miRNA-143-3p target sites in the 3\({}^{\prime}\)-UTR of _PCSK7_ are functional. No significant alteration was found in the relative luciferase activity in cells transfected with the miR-143-3p-overexpressing vector and Mut-a,b in the 3\({}^{\prime}\)-UTR of the psiCHECK-2 vector, in which both miR-143-3p target sites were deleted. However, the relative luciferase activity significantly diminished following the co-transfection of the miR-143-3p-overexpressing vector and Mut-a,b in the 3\({}^{\prime}\)-UTR of the psiCHECK vector, in which only one target site was absent. (**C**) No significant changes were observed in luciferase activity after the deletion of the only target site of miR-409-3p in the 3\({}^{\prime}\)-UTR of _PCSK7_. Mut-a stands for the deletion of the first target site, Mut-b stands for the deletion of the second target site, and Mut-a,b stands for the deletion of both miRNA target sites. \({}^{\star}\) and \({}^{\star}\) stand for \(p\) value less than 0.05 and 0.01, respectively. C1) and miR-224-5p controls (Figure 6A,B). As a control, a human PCSK9-overexpressing vector was co-transfected with miR-125a-5p and miR-224-5p vectors in Huh7 cells, and these miRNAs had no effect on the expression level of PCSK9 protein (not shown). Next, Huh7 cells were co-transfected with vectors expressing PC7 and miR-125a-5p at DNA ratios of 1:2 (2X), 1:3 (3X), and 1:5 (5X). The results showed that all three ratios of miR-125a-5p significantly decreased PC7 protein levels by >80%, with no significant PC7-silencing differences between the different amounts of miR-125a-5p (Supplementary Figure S3). We previously reported that PC7 specifically cleaves the human type-II membrane-bound hTfR1 into a soluble secreted form, thereby enhancing its circulating levels [8]. In addition, our bioinformatics analysis based on the UCSC genome browser (miRcode predicted microRNA target sites track) demonstrated that miR-125a-dp does not have any target sites on the 3'-UTR of hTfR1. In agreement with this fact, miR-125a-5p that reduced the level of the PC7 protein (Figure 6, Supplementary Figure S3) exerted no effect on the expression level of the endogenous hTfR1 protein in Huh7 cells (Supplementary Figure S4). Figure 6: Western blot analysis of PC7 in Huh7 cells, in which miR-125a-5p and miR-224-5p were overexpressed. (**A**) The expression of PC7 protein was significantly diminished by miR-125a-5p at 48 h post-transfection compared to mock counterpart vectors, whereas miR-224-5p did not change the protein levels of PC7.