Study design, subjects and sampling
1056 HCC patients and 1,080 healthy individuals were included in this retrospective cohort in Chinese Han population collected form archives department of Qingdao Sixth People's Hospital from May 2008 to July 2015. The numbers of male and female HCC patients and healthy individuals were approximate equal. Patients with cancer history were excluded. This study was approved by the ethics committee of Qingdao Sixth People's Hospital (ethics code: QDPHCAN132×1P). All patients were asked to provide 5 ml venous blood and were required to write informed consent with signature.
Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)
Total RNA was extracted from HCC tissues and adjacent normal tissues using an RNeasy Mini kit (Qiagen, Inc., Valencia, CA, USA), according to the manufacturers' protocol. RNA was reversed transcribed using a PrimeScript RT Master Mix kit (Takara Bio, Inc., Otsu, Japan). All forward and reverse primers were synthesized by Invitrogen (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The sequences were as follow: AKT1, Forward: 5′-CTTCCTCACAGCCCTGAAGTAC-3′, Reverse: 5′-GCATGAGGTTCTCCAGCTTGAG-3′; GAPDH, Forward: 5′-CCAGGGCTGCTTTTAACTCTG-3′, Reverse: 5′-CGCTCCTGGAAGATGGTGATG-3′. For amplification diluted cDNA was combined with a reaction mixture containing SYBR-Green PCR core reagents (cat. no. 4304886; Applied Biosystems; Thermo Fisher Scientific, Inc.). Relative mRNA expression levels were calculated using the 2−ΔΔCq method (19). PCR cycling was performed under the following conditions: 94°C for 30 sec and 45 cycles of 95°C for 5 sec, 56°C for 10 sec and 72°C for 10 sec. The results were expressed as the n-fold of the control.
Migration assay
The migration of HCC cells were evaluated using transwell plates. The isolated HCC cells were directly seeded on the upper chamber (8 mm pore size, 6.5 mm diameter; Corning Incorporated, Corning, NY, USA) with DMEM (Thermo Fisher Scientific, Inc.). The lower chamber was filled with DMEM (Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Thermo Fisher Scientific, Inc.). Cells were then incubated at 37°C for 48 h. The migrated cells from at least six random microscopic fields (×200) were counted under a light microscope (Olympus Corporation, Tokyo, Japan).
Western blot analysis
HCC tissues and adjacent normal tissues were homogenized in a lysate buffer containing protease-inhibitor (P3480; Merck KGaA, Darmstadt, Germany) and were centrifuged at 6,000 × g at room tempreture for 10 min. Western blot analysis was subsequently performed as previously described (20). Protein concentration was measured by a BCA protein assay kit (Thermo Fisher Scientific, Inc.). Protein samples (10 µg/lane) were resolved by 12.5% SDS-PAGE and then transferred to polyvinylidene fluoride membranes (Merck KGaA). After blocking with 2% bovine serum albumin (Sigma-Aldrich; Merck KGaA), rabbit anti-human AKT1 (ab81283) or GAPDH (ab9485) antibodies (all, 1:2,000; Abcam, Shanghai, China) were incubated with protein samples for 2 h at room temperature. Membranes were washed with PBS for 15 min at room temperature and then incubation with horseradish peroxidase-conjugated polyclonal anti-rabbit immunoglobulin G antibodies (1:10,000; PV-6001; OriGene Technologies, Inc., Beijing, China) for 1 h at room temperature. Signals were visualized by chemiluminescence detection (Z370398; Merck KGaA). Densitometric quantification of the immunoblot data was performed using Quantity-One software (v3.24; Bio-Rad Laboratories, Inc., Hercules, CA, USA).
Immunohistochemistry
Immunohistochemical procedures were performed as described previously (21). HCC tissues and adjacent normal tissues were frozen and coronal sections were cut in a cryostat. The tissues were cut into 4-µm thick sections and mounted on glass slides. The paraffinized sections were heated in an oven at 65° for 24 h, dewaxed to water and rinsed with PBS three times. The washed sections were placed in EDTA buffer (Beinuo Bioscience Inc., Shanghai, China), and then boiled at a low heat following an interval of 10 min at 65°C for a total of 3 intervals. Following natural cooling, the sections were washed with PBS three times, and were placed into 3% hydrogen peroxide solution (Beina Bioscience Inc.), for incubation at room temperature for 10 min, to block endogenous peroxidase. Free-floating sections were rinsed with PBS and placed in a solution containing primary mouse monoclonal antibodies directed against AKT1 (ab81283, 1:2,000; Abcam) at 4°C overnight. After rinsing with PBS, sections were incubated for 1 h at room temperature with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG mAb (1:5,000 dilution; PV-6001, OriGene Technologies, Inc., Beijing, China). The sections were then washed with PBS and observed by fluorescent video microscopy (BZ-9000; Keyence Corporation, Osaka, Japan).
DNA genotyping
All candidates' loci of AKT1 gene for tag gene were based on NCBI dbSNP database and SNP info. The genomic DNA was 10 µg of genomic DNA were isolated from extracted by the method of buffy-coat fractions with TIANamp blood DNA kit (Tiangen Biotech Co., Ltd., Beijing, China) (50 ng of genomic DNA, 200 µM dNTP, 2.5 units of Taq DNA polymerase, and 200 µM primers) and used for PCR amplification followed preliminary denaturation at 94°C for 2 min, followed by 35 cycles of 94°C for 30 sec, annealing temperature reduced to 64°C for 30 sec, and 72°C for 10 min by volume of 20 µl containing 50 ng of genomic DNA, 200 µM dNTP, 2.5 units of Taq DNA polymerase, and 200 µM primers. PCR primers (Forward: 5′-CTTCCTCACAGCCCTGAAGTAC-3′, Reverse: 5′-GCATGAGGTTCTCCAGCTTGAG-3′) were designed using Sequenom Assay Design v3.1 software (Sequenom, San Diego, CA, USA). Genotyping of AKT1 gene was conducted by PCR-restriction fragment length polymorphisms (RFLP) as described previously (22,23).
Trans-well invasion assay
HCC tissues were obtained from Qingdao Sixth People's Hospital. Transwell invasion assays were carried out in 24-well plates. In brief, HCC cells in serum-free medium contained Matrigel insert filters at 1:6 dilutions. The lower chamber was filled with culture medium with 10% FBS+DMEM. After incubation for 48 h at 37°C, cells were fixed and stained with 0.1% crystal violet (Sigma-Aldrich; Merck KGaA). The cells that invaded through the Matrigel membrane were quantified.
Luciferase assays
HCC cells were cultured in a 24-well culture plates for 24 h. Each well was transfected with 1.0 µg of each AKT1-reporter plasmid (pRL-SV40) with the allele C or T using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). The control-reporter vector was used as a negative control. HCC cells were transfected with 1.0 µg pRL-SV40 (containing the Renilla luciferase gene) plasmids per well. HCC cells were then lysed with the passive lysis buffer (Promega Corporation, Madison, WI, USA) and for luciferase expression activity analysis using the Dual-Luciferase Reporter Assay System (Promega Corporation) after 48-hour transfection. Three independent transfection experiments were performed in this experiment.
Statistical analysis
Continuous variables were shown as mean ± SD and analyzed by student t test. All data were analyzed using SPSS Statistics 19.0 and Graphpad Prism v5.0 with the help of Microsoft Excel. Allele and genotype frequencies were calculated by using direct counting. Hardy-Weinberg equilibrium (HWE) and the differences between allele and genotype frequencies were calculated using Fisher's exact test. Results of allele and genotype frequencies were determined by STATA SE 12.1 software. The risk of HCC was analyzed by regression analysis. *P<0.05 was considered to indicate a statistically significant difference.