Quantitative reverse transcription-polymerase chain reaction (qRT-PCR)

For each qRT-PCR experiment, 1 μl of diluted cDNA was used as the template. Quantification of mature miRNAs was performed using Maxima SYBR Green/ROX qPCR Master Mix (Thermo, Eugene, OR) in triplicate 20 μl reactions according to the manufacturer’s protocol with an Applied Biosystems 7500 real-time PCR system. Thermal cycling was organized into 2 steps: a first denaturation step of 10 min at 95°C, followed by 40 repeated cycles of 95°C for 10 sec and 60°C for 31 sec. U6 snRNA was used as the endogenous control for tissues, cells, and the medium. qRT-PCR forward primers for miRNA were ordered from Guangzhou RiboBio Co., Ltd. (Guangzhou, China). The qRT-PCR reverse and forward primers for U6 snRNA were from a miScript SYBR Green PCR Kit (Qiagen, Hilden, Germany). The expression level of each miRNA was normalized to that of the control. The ΔΔCt method was used to measure the expression level of each miRNA. ΔCt = Ct (target)− Ct (control). ΔΔCt = ΔCt (tumor)– ΔCt (non-tumor) or ΔΔCt = ΔCt (HCC cell line)– ΔCt (L02). The fold change of each miRNA was calculated using the 2^−ΔΔCt method.