## 2 Results
### Direct Interaction between the Predicted miRNAs and the 3'-UTR of PCSK7  
A dual-luciferase assay was applied to investigate the interaction between the predicted miRNAs and the 3'-UTR of human _PCSK7_ mRNAs, cloned downstream of the _Renilla luciferase_ gene in the psiCHEK-2 plasmid. Additionally, miR-224-5p without any target site on the 3'-UTR of _PCSK7_ served as a control. Here, the overexpression of miR-125a-5p significantly reduced the relative luciferase activity by \(\sim\)50% (\(p\) < 0.0001) in HEK293T cells co-transfecteded with the miR-125a-5p-overexpressing plasmid and a psiCHECK-2 carrying the wild-type _PCSK7_3'-UTR (Figure 4). Likewise, miR-143-3p and miR-409-3p significantly decreased the luciferase activity by \(\sim\)20-30% by targeting the _PCSK7_3'-UTR (\(p\) = 0.0013 and \(p\) = 0.04, respectively). However, miR-320a-3p and miR-224-5p, used as controls, had no significant effect (\(p\)\(\geq\) 0.05 and \(p\) > 0.99) on luciferase activity. Accordingly, miR-125a  
Figure 2: qPCR analysis of the expression levels of _PCSK7_ mRNA and predicted microRNAs in three examined cell lines: HEK293T, HepG2, and Huh7. The expression level of _PCSK7_ mRNA is the highest in Huh7, whereas miR-143-3p and miR-409-3p have the lowest expression in all the examined cell lines. 5p, miR-143-3p, and miR-409-3p were chosen for further analysis of their possible direct interactions with the _PCSK7_ 3'-UTR. Another set of luciferase assays in HEK293T and HepG2 cells were performed to confirm the interaction of miR-125a-5p (Supplementary Figure S1B). The 3'-UTR (whole is 926 bp) of the _PCSK7_ were divided into two parts, the proximal segment (647 bp, UTR-1) and the distal segment (275 bp, UTR-2), and separately cloned downstream of the _Renilla luciferase_ gene in the psiCHEK-2 plasmid (Supplementary Table S1). The experiments were performed in three biological replicates. As indicated in Figure 1, miR-125a-5p has one target site in each segment. Briefly, each miR-overexpressing version vector was co-transfected with each UTR constructs (Wildtype 3'-UTR, 3'-UTR-1, and 3'-UTR-2) in HEK293T and HepG2 cells. Forty-eight hours after transfection, the dual-luciferase assay was applied. Similar effects on _PCSK7_ UTRs were observed in both cell lines (Supplementary Figure S1B). Here, the overexpression of miR-125a-5p significantly reduced the relative luciferase activity in the HEK293T cells and HepG2 cells, co-transfected with the miR-125a-5p-overexpressing plasmid and a psiCHECK-2 carrying the wild-type _PCSK7_ 3'-UTR, the first part of _PCSK7_ 3'-UTR (UTR-1), and the second part of _PCSK7_ 3'-UTR (UTR-2). Likewise, miR-125a-5p targeted both UTR-1 and UTR-2 and the wild-type _PCSK7_ 3'-UTR in both transfected cell lines: in HEK293T cells (\(p\)-values were \(p\)\(<\) 0.0001, \(p\)\(=\) 0.0043, and \(p\)\(<\) 0.0001, respectively) and in HepG2 cells (\(p\)\(<\) 0.0001, \(p\)\(=\) 0.0247, and \(p\)\(<\) 0.0001, respectively). Both UTR-1 and UTR-2 harbored one target site of miR-125a-5p (Figure 1). Figure 3: Expression levels of _PCSK7_ after miRNA overexpression in Huh7 (**A**) and HEK293T (**B**) cells compared with their mock vector counterpart. A,B) The expression level of _PCSK7_ mRNA in Huh7 cells was significantly downregulated by miR-143-3p and miR-409-3p. In both Huh7 and HEK293T cell lines, miR-125a-5p downregulated _PCSK7_ expression, although the difference did not reach statistical significance in Huh7 cells (\(p\)\(=\) 0.06). In addition, miR-320a-3p could not downregulate the expression of _PCSK7_ in either Huh7 or HEK293T cells. * and ** stand for \(p\) value less than 0.05 and 0.01, respectively. The effects of the combination of predicted microRNAs (mix microRNAs) on PC7 expression were assessed via co-transfection of the combined the miRNAs and 3\({}^{\prime}\)-UTR of the _PCSK7_. However, the fold change in the luciferase activity did not vary significantly compared to the effect of each individual microRNAs (Supplementary Figure S2).