Tissue specimens and tissue microarray (TMA)
mRNA and protein samples of human HCC tissues (n=48) and matched adjacent nontumor samples (n=48) were collected from patients who had undergone curative hepatectomy at Zhongshan Hospital Affiliated with Fudan University.
Preparation of TMA was performed as described. Primary HCC samples for TMA were obtained from 241 postoperative patients with HCC whose 10-year follow-up data were available at Zhongshan Hospital Affiliated with Fudan University. The study was approved by the Zhongshan Hospital Research Ethics Committee. Follow-up procedures were described previously.
The slides were probed with primary antibodies against PCSK9 (1:100, Abcam, Cambridge, MA, USA) and then incubated with horseradish peroxidase–conjugated IgG (1:500; Invitrogen), and the proteins in situ were visualized with 3,3’-diaminobenzidine. The staining extent was determined as previously reported.
The human HCC cell lines HepG2 and Huh7 were purchased from the Cell Bank of the Institute of Biochemistry and Cell Biology, China Academy of Sciences, Shanghai, China. The HCC cell lines MHCC-97H and MHCC-97L were established at the Liver Cancer Institute. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (Gibco, BRL, USA), 10 µg/mL streptomycin sulfate and 100 µg/mL penicillin G. All cells were incubated at 37°C in a humidified atmosphere containing 5% CO2.
Total RNA isolation and quantitative real-time quantitative PCR
Total RNA was isolated from HCC cells and HCC tissues with TRIzol reagent (Invitrogen, CA, USA) according to the manufacturer’s protocol. Complementary DNA was synthesized using the Reverse Transcription Reagent Kit (TaKaRa, Shiga, Japan) from 500 ng of RNA. All primers are shown in Supplementary Table 1. Real-time qRT-PCR analysis was performed using an ABI7500 real-time fluorescent measurement system (Applied Biosystems, USA) and a PCR amplification kit (TaKaRa, Japan). Values were derived from at least three independent experiments performed in duplicate and normalized to β-actin expression. The 2-△△Ct method was used for the data analysis.
The procedures were performed as described. Preparation of nonreducing PAGE gel and protein sample was as follows: preparation of the nonreducing gel was according to the second part of the concentrated rubber and separation gel preparation scheme, without adding SDS. After the protein was extracted, it was diluted with 5× nondenaturing and nonreducing buffer in a 4:1 ratio without boiling and directly added to the sample hole of the nonreducing PAGE gel. The antibodies used for western blotting included PCSK9 and GSTP1 (Abcam, Cambridge, MA, USA).
Cell viability assays
HCC cell viability was assessed using CCK-8 assays (Cell Counting Kit-8, Dojindo, Japan). The procedures were performed as described.
Cell cycle analysis and Annexin V apoptosis assay
The procedures were performed as described.
Coimmunoprecipitation (Co-IP) and mass spectrometry (IP/MS)
The procedures were performed as described[10, 15]. The antibodies used in Co-IP included anti-PCSK9 mAb, anti-GSTP1 mAb, and anti-IgG (Abcam, Cambridge, MA, USA).
HCC cells grown on coverslips were fixed with 3% paraformaldehyde at 4°C overnight. To block the nonspecific binding of antibodies, cells were then incubated with 3% bovine serum albumin for 30 min at room temperature. After that, cells were incubated with primary antibodies against PCSK9 (1:100, Abcam, Cambridge, MA, USA) and GSTP1 (1:100, Abcam, Cambridge, MA, USA) at 4°C overnight. Cells were then probed with secondary antibodies and incubated at room temperature for another 2 h. Cells were then mounted with ProLong gold antifade reagent with 4',6-diamidino-2-phenylindole (Life Technologies) and immediately observed under a fluorescence microscope.
Nude mice and metastasis assay
HCC cells (1.2 × 108 cells in 0.1 ml of phosphate-buffered saline) were injected subcutaneously into the dorsal left flank of eight-week-old male Balb/c nude mice. These mice were randomly divided into four groups before injection. After the tumors were visible, the tumor size was measured every week until 4 weeks. The nude mice were killed, and the subcutaneous tumor was stripped and cut into tumor tissue blocks of 1 mm × 1 mm × mm size as the tumor source for subsequent orthotopic liver planting. The mice inoculated with tumor blocks were sacrificed after eight weeks, and the number of metastatic tumors was assessed by double-blinded evaluation.
Statistical analysis was performed with SPSS 19.0 statistical software (SPSS Inc., Chicago, IL, USA). Kaplan-Meier analysis was used for survival analysis, and the log-rank test was chosen to compare the differences. The Pearson χ2 test or Fisher’s exact test was employed to compare qualitative variables, while Student’s t-tests were used for quantitative variables. A Cox proportional hazards model was adopted for multivariate analysis. The level of significance was set at P<0.05 for all tests.