## 4 Materials and Methods
### Cell Culture  
Human embryonic kidney 293 (HEK293T), hepatocellular carcinoma (Huh7), and hepatocellularoma (HepG2) cells were selected for further functional experiments. The HEK293T and Huh7 cell lines were cultured in Gibco Dulbecco's Modified Eagle Medium (DMEM) (Invitrogen, Headquarters Thermo Fisher Scientific, Waltham, MA USA), supplemented with 100 U/mL of penicillin, 100 ug/mL of streptomycin (Sigma, Massachusetts Millipore, Spring, Burlington, MA USA), and 10% fetal bovine serum (FBS) (Invitrogen, Headquarters Thermo Fisher Scientific, Waltham, MA USA); then, they were incubated at 37 \({}^{\circ}\)C with 5% CO\({}_{2}\). The HepG2 cells were cultivated in DMEM-F12 (Invitrogen, Headquarters Thermo Fisher Scientific, Waltham, MA USA), containing 10% FBS, 100 U/mL of penicillin, and 100 ug/mL of streptomycin.  
RNA Extraction, Complementary DNA (cDNA) Synthesis, and Quantitative Polymerase Chain Reaction (qPCR)  
RNA was isolated from the cells with the TRIzol Reagent (Invitrogen, Waltham, MA, USA) according to the manufacturer's instructions. The RNA samples were treated with RNase-free DNase I (Fermentas, Waltham, MA, USA) to eliminate any possible DNA contamination. Reverse transcription was performed using the PrimeScript First Strand cDNA Synthesis Kit (Takara, Kusatsu, Japan) following the manufacturer's protocol. Briefly, one unit of the DNase I enzyme, 1 uL of a buffer, and 1 ug of total RNA were incubated for 30 min at 37 \({}^{\circ}\)C. Then, 1 uL of 50 mM EDTA was added for enzyme inactivation and incubated at 65 \({}^{\circ}\)C for 10 min. Subsequently, 5 uL of DNase-treated RNA was added to the mix of 0.5 uL of the reverse-transcriptase enzyme, 2 uL of the reverse-transcriptase buffer, and 1 uL of a random hexamer and incubated for 15 min at 37 \({}^{\circ}\)C, followed by 5 s at 85 \({}^{\circ}\)C for enzyme inactivation. The stem-loop RT-qPCR method was applied for evaluating the expression of miRNAs [50].  
The qPCR test was conducted in 20 uL of the PCR reaction mixture using SYBR Green I (Takara, Kusatsu, Japan) in an Applied Biosystems StepOne instrument (Applied Biosystems, Waltham, MA, USA). Briefly, cDNA equivalent to 50 ng of RNA was added to the mix of 10 uL of SYBR Green, 0.5 uM of each primer, 0.4 uL of the ROX reference dye, and sufficient water. The real-time thermal program was as follows: 95 \({}^{\circ}\)C for 30 s, 40 cycles at 95 \({}^{\circ}\)C for 20 s, and 60 \({}^{\circ}\)C for 35 s for _PCSK7_, as well as 95 \({}^{\circ}\)C for 30 s, 40 cycles at 95 \({}^{\circ}\)C for 5 s, 60 \({}^{\circ}\)C for 30 s, and 72 \({}^{\circ}\)C for 15 s for miR-125a-5p, miR-143-3p, miR-409-3p, miR-320a-3p, and miR-244, for which RNU48 small nuclear RNA, _\(\beta\)2-microglobulin (\(\beta\)2m)_, and _GAPDH_ mRNAs were used as internal controls.  
All the reactions were repeated in duplicates. Next, melt curves were analyzed, with the mean threshold cycles used for further analyses. The relative expressions of the miRNAs and _PCSK7_ to RNU48 small nuclear RNA and/or _GAPDH_ and _\(\beta\)2m_ were calculated, respectively, via the 2\({}^{-\Delta\Delta\text{CI}}\) method. All expression experiments were performed in three biological replicates.  
The PCR products were sequenced (3500 ABI) to validate the accuracy of the amplification. All the primers for _PCSK7_ and the nominated miRNAs are listed in Supplementary Table S1.