## 2 Results
### Expression of the Predicted miRNAs and PCSK7 in HEK293T, HepG2, and Huh7 Cells  
We next used qPCR to evaluate the endogenous expression patterns of the four selected miRNAs (miR-125a-5p, miR-143-3p, miR-409-3p, and miR-320a-3p) and _PCSK7_ mRNA in three cell lines, namely kidney-derived HEK293T, hepatocytes-derived HepG2, and Huh7 cells (Figure 2).  
Huh7 cells showed the highest relative expression of _PCSK7_ mRNA and extremely low RNA expression levels of miR-143-3p and miR-409-3p. The expression levels of miR-125a-5p and miR-320a-3p were similar in all cell lines. In HepG2 and HEK293T cells, the expression levels of miR-125a-5p and miR-320-3p were comparable to that of _PCSK7_, while the expression levels of miR-143-3p and miR-409-3p were low. From these data, it seems that the levels of _PCSK7_ mRNA are inversely correlated to the RNA levels of miR-143-3p and miR-409-3p in all cells, whereas miR-125a-5p may negatively regulate _PCSK7_ mRNA levels in Huh7 cells.  
Figure 1: Schematic view of the genome organization of PCSK7. (**A**) Exons, introns, and miRNA target sites on the PCSK7 sequence. Notice the presence of two putative sites targeted by miR-125-5p on exons 14 and 15 (red lines). (**B**) The target sequences of miRNAs in the 3\({}^{\prime}\)-UTR of PCSK7 and their nucleotide hybridization status. Note that miR-125-5p (red a and b) and miR-143-3p (violet a and b) target two sites in the 3\({}^{\prime}\)-UTR. The target sites of miR-409-3p and miR-320a are presented as blue and green colors, respectively.  
Negative Regulation of PCSK7 mRNA Expression Levels by the Overexpression of miR-125a-5p, miR-143-3p, and miR-409-3p  
Huh7 cells were transfected with miR-overexpressing vectors carrying the precursors of miRNA sequences and mock-related counterpart vectors to assess the possible relationship between the expression patterns of miR-125a-5p, miR-143-3p, miR-409-3p, and miR-320a-3p with _PCSK7_ expression at the transcriptional level. The qPCR results obtained at 48 h following transfection demonstrated that the overexpression of miR-143-3p and miR-409-3p in Huh7 cells significantly decreased the expression of _PCSK7_ mRNA by 40-50% compared to cells transfected with the mock control vector (\(p\) = 0.0158 and \(p\) = 0.0084) (Figure 3A). Although the overexpression of miR-125a-5p tended to reduce the expression of _PCSK7_ mRNA in these cells, this decrease did not reach statistical significance (\(p\) = 0.0693). In contrast, the ectopic expression of miR-320a-3p did not downregulate the expression of _PCSK7_ in Huh7 cells (\(p\) = 0.5538).  
The association between the expression levels of miR-125a-5p and miR-320a-3p and the expression level of _PCSK7_ was further investigated by measuring the levels of _PCSK7_ mRNAs in two other cell lines, namely HEK293T and HepG2 cells. The qPCR results showed that the overexpression of miR-125a-5p now led to a significant \(\sim\)25% reduction in _PCSK7_ mRNA levels 48 h after transfection in these cells (\(p\) = 0.0114, \(p\) = 0.0010, respectively) (Figure 3B and Supplementary Figure S1A). However, like Huh7 cells, no significant regulatory relationship was observed for miR-320a-3p in HEK293T (\(p\) = 0.9972) and HepG2 (\(p\) = 0.7830) cells. We conclude that the overexpression of miR-143-3p, miR-409-3p, and less so miR-125a-5p may downregulate _PCSK7_ expression.