Quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) assay

The HCC and adjacent normal tissue samples were retrieved from liquid nitrogen and grinded to tiny particles, before incubation with TRIzol (Takara, Otsu, Shiga, Japan) to isolated mRNA. HepG2 cells that were harvested in 35 mm dishes were also incubated with TRIzol (Takara) to extract mRNA. 500 ng RNA was applied to synthesis first-strand complementary DNA using first-strand cDNA Synthesis Kit (Takara, Otsu, Shiga, Japan). RT-PCR was performed in a mixture composed of SYBR Green (Takara), 1 μl (0.2 μmol/l) of each primer, and 1.6 μl of complementary DNA from RT-PCR samples. The primer sequences were as follows: AEG-1, 5'-CGGTACCCCGGCTG GGTGAT-3' (forward) and 5'-CTCCTCCG CTTTTTGCGGGC-3' (reverse); MDR-1, 5'-GCCGGGAGCAGTC ATCTGTGG-3' (forward) and 5'-ATCCAT TCCGACCTC GCGCT-3' (reverse); GAP DH, 5'-CAAT GACCCCTTCATTGACC-3' (forward) and 5'-GACAAGCTTCCCGTTC TCAG-3' (reverse). The mRNA levels of AEG-1 and MDR-1 were quantified by the Ct values, and GAPDH was set as an internal control.

Experiments were performed in triplicate. The relative expression levels were evaluated using the ^-ΔΔCt method as described previously (Yang et al., 2014[29]). Each individual experiment was performed three times independently.