Three fresh frozen tissue samples were used: an FLHCC in a 33-year-old male patient with multiple hepatic tumors and diffuse peritoneal carcinomatosis that developed severe and refractory hyperammonemic encephalopathy (ammonia blood level: 312 mcml/L (reference interval: 9-30 mcmol/L)), a non-tumor non-cirrhotic liver (ammonia blood level: 26 mcmol/L) and a rapid growing hepatic adenomyomatosis in a 44-year-old female patient that developed hyperammonemia (ammonia blood level: 26 mcmol/L) submitted to percutaneous ultrasonography-guided diagnostic hepatic lesion biopsy. All tissue samples were obtained by Quick-Core needle biopsies (Cook Group Inc. Bloomington, Indiana). Samples were immediately submitted to cryopreservation with liquid nitrogen.
Reagents
TRizol (Invitrogen. Carlsbad, California. USA), diethylpyrocarbonate (DEPC), 2x SYBR Green Reaction Mix (Invitrogen), Super Script III RT/Platinum Taq Mix (Invitrogen), ROX Reference Dye (Invitrogen), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), beta-2 microglobulin (B2M), ethylenediaminetetraacetic acid (EDTA), and ethidium bromide solution (EtBr).
Real-time PCR (RT-PCR)
RT-PCR was used to determine messenger RNA (mRNA) levels of proteins of interest. Total RNA was extracted from 100 mg of frozen liver with TRIzol reagent following the manufacturer's instructions. RNA was dissolved in DEPC-treated water and quantified spectrophotometrically at 260 nm. One hundred nanograms of total RNA was used for each real-time PCR reaction, which was performed in StepOne equipment (Applied Biosystems Inc. Foster City, California). RT-PCR was performed in a 15 microl reaction mixture containing 7.5 microl 2x SYBR Green Reaction Mix, 0.3 microl each primer, 0.3microl Super Script III RT/Platinum Taq Mix (10 pmol/microl), 0.15microl ROX Reference Dye, and 5microl sample in water. cDNA synthesis was performed at 50oC for 15 minutes followed by 35 cycles at 95oC for 15 seconds, annealing temperature for 30 seconds, and 72oC for 30 seconds. Quantification was performed by the 2-DDCT method using GAPDH and B2M as housekeeping genes. The primer sequences and annealing temperature are listed in Table 1. Primers were designed using GeneRunner Software (Hastings Software Inc. Hastings, NY). The DNAJB1-PRKACA fusion transcript primer sequence was obtained by Graham et al. (Table 1).
Agarose gel electrophoresis
PCR amplicons fragments were separated via 1% agarose gel electrophoresis. The gels were prepared by dissolving the agarose in the TBE buffer (100 mM Tris, 100 mM boric acid, 2 mM EDTA). To visualize the DNA, 1.5 microl of (10 mg/ml) EtBr was added to the 100 ml 1% agarose solution. To load the samples, the DNA was mixed in equal volume ratios with the agarose gel loading buffer. Electrophoresis was performed at 100 V. The DNA was detected using UV light and the size of the DNA was determined using the standard 100bp DNA ladder.
Compliance with ethical standards
Tissue samples used in this research were obtained from already available biological material and patients signed an informed consent form before the use of the samples. Ethics clearance was obtained from the Ethics Research Committee at the University of Sao Paulo Medical Faculty and all experiments were performed in accordance with relevant guidelines and regulations.
Results
The DNAJB1-PRKACA fusion protein and mutation on chromosome 19 were detected only in the FLHCC sample (Figure 1).
The relative expression versus Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) of AURKA in FLHCC and adenomyomatosis samples presented increased expression of AURKA, c-Myc, and ornithine decarboxylase (Figure 2) when compared to normal liver. On the other hand, ornithine transcarbamylase was decreased in FLHCC and adenomyomatosis when compared to a non-tumor liver (Figure 3). There was no difference between FLHCC and adenomyomatosis in the expression of AURKA and c-Myc.
Samples analyses were performed by RT-PCR in triplicates. The results are expressed as relative expressions compared to GAPDH using the 2-DDCT method.