HCC tissue samples
From 2005 to 2014, tumor samples and corresponding adjacent normal tissues were collected from 92 HCC patients receiving surgery at the Affiliated Baiyun Hospital of Guizhou Medical University. The corresponding adjacent normal tissue samples were obtained > 5 cm from the edge of the cancerous region and there were no obvious tumor cells evaluated by pathologist. These tissue samples were conserved in liquid nitrogen after collection or prepared in paraffin sections. No systemic or local treatment had been received before operation. Both tumor and nontumor tissues were histologically confirmed. All the tissue samples were obtained with informed consent from all the patients. This study was approved by the Institute Research Ethics Committee of Guizhou Medical University.
Cell lines
HCC cell lines MHCC97L, Huh7, HepG2, HCCLM3, SMMC-7721, MHCC97H and normal liver cell lines HL-7702 were from the tumor cell bank of Chinese Academy of Sciences. All the cell lines were grown in Dulbecco’s modified eagle medium supplemented with 10% fetal bovine serum, 100 Ag/AL streptomycin, and 100 Ag/AL penicillin (pH 7.2–7.4) in a humidified incubator containing 5% CO2 at 37 °C.
Immunohistochemistry
For each patient sample, three paraffin sections of 5 μm were prepared, for immunohistochemical staining. Sections were dewaxed using xylene, followed by hydration with ethanol solutions and addition of EDTA for antigen retrieval. Later, sections were blocked with normal goat serum for 30 min to eliminate non-specific binding. Sections were incubated with primary antibody against PIK3R1 (Abcam, Cambridge, UK). Sections were then incubated with biotin-labeled secondary antibodies for 30 min at room temperature, followed by staining with diaminobenzidine (DAB).
Reverse transcription-quantitative PCR
Total RNA of tissues or cultured cells was isolated by using TRIzol reagent (Invitrogen). Total RNA (1 μg) was transcribed into cDNA by using a First-strand cDNA Synthesis System (Invitrogen). 1 μl DNA template was used to amplify by using Power SYBR® Green PCR Master Mix (ABI, USA) on the 7500 real time PCR system (ABI, life technology). The reaction system was performed in a volume of 20 μl. The GAPDH was used as a loading control for each specific gene. Each experiment was performed three times and each sample was tested in triplicate. The sequences of human PIK3R1 primers were 5′-TAGCTCGCGCGATCTAGGGGC-3′ (sense) and 5′-CGCGATCAATAAAGCTAG-3′ (antisense). The primers for human GAPDH were 5′-GCACCGTCAAGGCTGAGAAC-3′ (sense) and 5′-TGGTGAAGACGCCAGTGGA-3′ (antisense).
Western blot analysis
Whole cells were lysed on ice in a lysis buffer (RIPA, Beyotime, Shanghai, China) with a protease inhibitor mixture cocktail (Roche, Switzerland). After centrifugation at 12,000 rpm for 30 min at 4 °C, the protein concentrations of supernatants in samples were measured by the BCA protein assay (Thermo scientific, Rockford, IL, USA). Equal amounts of protein (30 μg) were separated by 10–12% NUPAGE Bis–Tris Gel (Invitrogen, CA, USA) electrophoresis (constant voltage: 120 mv) and transferred onto polyvinylidene fluoride (PVDF, 0.45 μm) membranes (constant current: 350 mA for 70/120 min). After being blocked by Tris-buffered saline and Tween 20 (TBST) buffer containing 5% non-fat powder milk for 2 h, the membranes were incubated with primary antibodies overnight on ice. After washing the membranes several times in TBST while agitating, detection was performed using the appropriate secondary HRP-conjugated anti-mouse or antirabbit antibody. Immunoreactive bands on the blots were visualized with enhanced chemiluminescence reagent ECL kit (Beit Haemek, Israel).
Small interfering RNA transfection of human HCC cell line
Human PIK3R1 siRNA (5′-CCTAGCGCATATCGCC-3′) and control-siRNA were synthesized by GenePharma (shanghai, china). Cells were transfected with sh PIK3R1 or control-shRNA using Lipofectamine 2000 (Invitrogen, Life Technologies), according to the manufacturer’s instructions.
MTT assays
The proliferation of cells was evaluated by the MTT assay. Cells were plated in a 96-well plate at 3 × 103 cells/well and were allowed to grow for different times. The growth rate was determined by the cell number and was counted in triplicate every day by MTT assay. Briefly, cells were incubated with 50 μl of 0.2% MTT for 4 h at 37 °C in a 5% CO2 incubator. Following MTT incubation, 150 μl of 100% DMSO was added to dissolve the crystals. Viable cells were counted every day by reading the absorbance at 490 nm using a 96-plate reader BP800 (Dynex Technologies).
Clone formation assay
Clone formation assay was conducted to examine the effect of PIK3R1-siRNA on cell growth in HCC cell lines. 4 × 105 cells were plated in a 6-well plate. After 24 h of transfection, the cells were trypsinized, and 1000 single viable cells were plated in three 6-well plates. The cells were then incubated for 14 days at 37 °C in the condition of 5% CO2. Colonies were stained with 0.1% crystal violet, washed with water, and counted ten random fields manually. The colonies containing at least 100 cells were scored. The surviving fraction in PIK3R1-siRNA transfected cells was normalized to untreated control cells with respect to clonogenic efficiency.
Wound healing assay
Wound healing assay was adopted to test the migration ability of HCC cells. In our study, cells were digested after transfection by specific siRNA and control siRNA to human PIK3R1 for 24 h in 6-well plates, 2 × 105 cells were plated in 24-well plates, when cell confluence reached approximately 100%, the old medium was removed and the monolayer was wounded by scratching with a 10-μl sterile pipette tip lengthwise along the chamber, then cells were washed three times with PBS and cultured with serum-free medium at 37 °C. Images of migrating cells into the wound were photographed at 0 h and 48 h using an inverted microscope. The scratch width of the cells was confirmed by detecting the width of the monolayer wound at 0 and 48 h, and the migration index was counted as follows: migration index = (0 h scratch width − 48 h scratch width)/0 h scratch width) × 100 [9].
Transwell migration assay
Cell migration ability was determined by transwell assays. The treated MHCC97H and HCCLM3 cells (1.0 × 105/ml) were seeded in the upper chambers (BD Biosciences, NY, USA). The upper chamber was filled with serum-free medium and the lower chamber was supplemented with 10% fetal bovine serum. Hence this allowed the cells in the upper chamber migrate into the lower chamber. After 48 h incubation, the cells that had invaded through the membrane were stained with 0.1% crystal violet solution. The sections were observed by using a light microscope (magnification at ×100).
Apoptosis assay
The apoptosis ability was measured by using Annexin V-FITC/PI apoptosis detection kit (BestBio, Shanghai, China). Cells were digested after transfection by specific siRNA and control siRNA to human PIK3R1 were washed with ice-cold PBS. The treated cells (1 × 106 cells/ml) were suspended with 100 μl 1 × binding buffer and double stained with Annexin V-FITC/PI for 15 min according to the manufacturer’s instructions. Intensities of fluorescence signals were measured on a FACS Calibur flow cytometer (Becton-Dickinson, Franklin-Lakes, NJ, USA). The image of apoptosis was divided into four quadrants: all living cells (double negative), early apoptotic cells (Annexin V-positive, propidium iodide-negative), necrotic cells (Annexin V-negative, propidium iodide-negative positive), as well as late apoptotic cells (double positive). We counted the early apoptotic cells and the late apoptotic cells.
Statistical analysis
For continuous variables, data are expressed as mean ± standard deviation (SD). The difference between PIK3R1 mRNA or protein expression in tumor tissue and that in adjacent normal tissues was evaluated using Student’s t-test. GraphPad Prism 5.0 Software was employed to perform statistical analysis. All statistical tests were two-tailed and statistical significance was assumed for p < 0.05.