Clinical tissues and tissue microarrays
HCC tissues and matched normal samples from The Affiliated Huai’an NO.1 People’s Hospital of Nanjing Medical University were used for western blot. Immunohistochemistry (IHC) analysis was performed on tissue microarray kits comprising 80 human HCC tissues and matched 80 healthy tissues that were procured from Outdo Biotech (Shanghai, China). Written informed consent was signed by patients and all experimental protocols were reviewed by the Ethics Committee of Nanjing Medical University.
Cell culture and RNA interference by shRNA
Six HCC cell lines and one normal liver cell line L02 were purchased from KeyGen (Nanjing KeyGen Biotech Co, Ltd, Jiangsu, China). Cells were maintained in 10% fetal bovine serum(FBS)-supplemented Dulbecco’s Minimum Essential Medium (DMEM) along with antibiotics in an atmospheric condition of 5% CO2 and at 37。C. Three siRNAs were purchased from Gene Pharma (China). PR55α target sequences utilized in this experiment were the following: GCCUAUGGAUCUAAUGGUUTT for siPR55α#1, GCAGAUGAUUUGCGGAUUATT for siPR55α#2, and GGAAACAUACCAGGUGCAUTT for siPR55α#3. Transfection was performed as previously documented18.
Western blotting assays were conducted as previously reported18. The primary antibodies are as follows PR55α(1/1000, A2185, Abclonal, China), AKT(1/1000, A17909, Abclonal, China), p-AKT-T308(1/1000, AP0304, Abclonal, China), P-AKT-S473 (1/1000, AP0140, Abclonal, China), ERK1/2(1/1000, A4782, Abclonal, China) and p-ERK1/2-T202/Y204(1/1000, AP0472, Abclonal, China).
The slices were deparaffinized in xylol, heated for antigen retrieval with sodium citrate (pH 6.0), and treated by hydrogen peroxide. Then, tissue microarray were incubated with anti-PR55α at 4℃ overnight. Finally, secondary antibody and DAB chromogentic agent were added. PR55α staining intensity was graded based on the following scale: 0 (staining not detectable), 1 (faintly yellow, weak staining), 2 (light brown moderately staining), and 3 (brown, strongly staining). A high PR55α expression was marked by colour grades of ≥2. The staining results were assessed by two senior pathologists that remained isolated from the other throughout the experiment.
Cell counting and colony formation assay
Colony formation assays and Cell Counting Kit-8 (CCK-8) assays were used to assess the ability of cell proliferation. 96-well plates were used to house transfected HCC cells at a density of 1×103 cells per well. After culturing overnight, the CCK-8 reagent (Dojindo, Shanghai, China) was added to the cells daily at the same time. Two hours later, a microplate reader was used to assess the absorbance values (OD).
Six-well plates were used to contain the transfected cells (1×103 cells per well) which was then allowed to undergo a 10-day incubation period. Colonies that obviously contained ≥50 cells were selected for counting. Phosphate buffered saline (PBS) was used to rinse the colonies before they were fixed for 30 minutes with 4% paraformaldehyde and exposed for 2 minutes to crystal violet. Three individual repeats of each experiment were done.
Cell migration and invasion assay
The invasive and migratory abilities of cells were measured by transwell assays. 200ul serum-free medium was used to resuspend 2×104 transfected HCC cells before they were placed in the upper cartridge (Milllicell, USA) which was precoated with or without 50ul matrigel. The lower cartridge contained 600ml DMEM medium with 20% FBS. The cells were then allowed to incubate for 24 hours. Paraformaldehyde was then used to fix cells before they were staining using crystal violet. Cells were then photographed and quantified using a microscope for data analysis. The results represented the mean values of three independent experiments.
Cell cycle and apoptosis assay
Cell cycle and apoptosis assays were conducted using flow cytometer (Beckman Coulter). For cell cycle analysis, transfected cells were harvested and fixed overnight in 70% ethanol at 4°C. The cells were then exposed to RNase A before being labeled for 30 minutes with propidium iodide (PI) at 37°C.
Cells were collected and stained with Annexin V-FITC and PI reagents prior to the cell apoptosis analysis. Three separate repetitions were carried out for each experiments.
In vivo tumor assay
BABL/c nude mice were obtained from Nanjing Medical University (Nanjing, Jiangsu, China). Six-week-old female nude mice (n=5) received subcutaneous injections of approximately 2x106 LM3 cells which possessed stable PR55α knockdown or PR55α control to their flanks. The xenograft tumors were dissected and imaged at 4 weeks post-inoculation. Terminal deoxynucleotidyl transferase (TdT) mediated dUTP nick end labelling (TUNEL) staining and Ki-67 immunofluorescence staining were performed as described previously18. For experiments regarding lung metastases, the same cancer cells were administered intravenously into the tail veins of nude mice (n=5). Lung nodules were measured under a dissecting microscope after 40 days of incubation. The Experimental Animal Management Committee of Nanjing Medical University approved all mice experiments which were formulated in strict compliance to established guidelines.
RNA sequencing analysis and quantification were utilized to assess changes in mRNA profiles of LM3 cells with PR55α knockdown or PR55α control. Samples were performed in triplicate. The differentially expressed mRNAs were selected with fold change < 0.5 or >2 and possessed a p value < 0.05 by R package edgeR. We also analyzed GO enrichment and KEGG enrichment in the differentially expressed mRNAs.
Mean ± standard deviation was used to express all collected data. Analysis was carried out using GraphPadPrism 6. Variances between normal and HCC samples were contrasted using Student’s t test. The associations between clinicopathological features and PR55α expression were assessed by Fisher’s exact test and chi-square test. Statistical significance was designated as follows: * P<0.05, ** P<0.01 and *** P<0.001. P<0.05 was interpreted as a result that was statistically significant.