Increased PADI4 expression in blood and tissues of patients with malignant tumors

Various malignant tumors, benign tumors and non-tumor inflamed tissues (n = 1673, see Table 1) were collected during excision surgery at several hospitals in the Shandong region of China. Tumor diagnosis was verified by histological methods, and pathological categorization was determined according to the World Health Organization (WHO) classification system. All patients signed informed consents, and this study was approved by the ethics committee of Shandong Academy of Medical Sciences.

Tissue samples were fixed in 10% neutral buffered formalin and embedded in paraffin. Tissue sections were deparaffinized and rehydrated using standard procedures. To increase the intensity of immunostaining, the sections were heated at 95°C for 10 min in citrate buffer (0.01 M, pH 0.6), then incubated overnight at 4°C with anti-PADI4 antibody. Anti-PADI4 was prepared by immunizing rabbits with the synthetic oligopeptide FGDSCYPSNDSRQMH, and immunospecificity was confirmed in a previous study [3]. Immunoreactions were processed using the UltraSensitive TM S-P Kit (Maixin-Bio, China) according to the manufacturer's instructions, and signals were visualized using the DAB substrate, which stains the target protein yellow.

The intensity of immunosignals was evaluated by a previously described protocol from Denkert et al. [8]. For each histological section, the percentage of positive cells was scored as 0 (0%), 1 (10%), 2 (10–50%), 3 (51–80%) and 4 (> 80%), and the staining intensity was scored as 0 (negative), 1 (weak), 2 (moderate) and 3 (strong). The immunoreactive score (IRS) was obtained by multiplying the percentage of positive cells and the staining intensity. Immunohistochemical results with an IRS of 0–1 were considered negative, 1–2 weak and 6–12 positive.

Gastric adenocarcinomas (n = 5), lung adenocarcinomas (n = 5), hepatocellular carcinomas (n = 4), esophageal squamous cell cancers (n = 5), breast cancers (n = 5), and breast fibroadenomas (n = 5) were collected during excision surgery. Normal, healthy tissues located 5 cm away from the corresponding tumors were collected during surgery for control histological examinations. Sample tissues (200 ug) were homogenized with Cell Lysis Solution (Sigma) and centrifuged at 16000 × g for 5 min at 4°C. The supernatant was collected, and protein concentrations were determined using the BCA Protein Assay Kit (Pierce). Immunoprecipitation (IP) was performed using a Protein G Immunoprecipitation Kit (Sigma) according to the manufacturer's instructions. Briefly, an equal amount of lysate from each sample was incubated overnight at 4°C with the PADI4 antibody in the presence of a Protease Inhibitor Cocktail (Sigma). Protein G beads were added to the mixture and incubated for 2 h at 4°C. After thorough washing, the purified PADI4 protein was eluted with 1× Laemmli sample buffer (Sigma). IP samples (10 ul) were separated by SDS-PAGE and then transferred onto PVDF membranes using the Mini-PROTEAN 3 system (Biorad). The membranes were probed with monoclonal anti-PADI4 antibody containing a recombinant fragment of human PADI4 between residues 2–111 (Abcam), and then incubated with sheep anti-mouse IgG conjugated to alkaline phosphatase (Sigma). Immunosignals were visualized with the Protein Detector BCIP/NBT Western Blot Kit (KPL) following the manufacturer's instructions.

We also measured PADI4 expression in total protein extracts from the tumor cell lines A549, SKOV3 and U937. Preparation of protein extracts was performed as described above.

Blood samples were collected from patients with malignant tumors 4–6 days prior to and after tumor excision surgery. Blood from healthy volunteers and from patients with benign tumors or non-tumor inflammation were used as controls. All samples (n = 1121, see Table 2) were collected in Monovette tubes containing 3.8% sodium citrate and centrifuged at 1500 × g for 20 min. Supernatants of anticoagulated plasma were collected and stored at -80°C until use. Plasma samples were diluted 1:20 in 0.05 M carbonate-bicarbonate buffer (pH 9.6) and used to coat 96-well EIA/RIA microplates (Costar) overnight at 4°C. After a brief wash with PBST (8 g NaCl, 0.2 g KCl, 1.15 g NaHPO₄ and 0.2 g KH₂PO₄ per liter, pH 7.4–7.6, 0.1% Tween 20), plates were blocked with 5% non-fat dry milk for 1 h at room temperature. Anti-PADI4 antibody (diluted 1:4000 in PBST) was added to the plates and incubated for 2 h at room temperature. After washing with PBST, plates were incubated with 1:10,000 anti-rabbit IgG conjugated to alkaline phosphatase (Sigma) for 30 min at room temperature. Plates were then washed with PBST, and the signal was developed by adding the Alkaline Phosphatase Yellow (pNpp) Liquid Substrate System for ELISA (Sigma). Absorbance at 405 nm was measured using a spectrophotometer (Synergy HT, Bio-Tek).

Anti-human antithrombin monoclonal antibody (Abcam) was diluted 1:5000 in 0.05 M carbonate-bicarbonate buffer (pH 9.6) and used to coat microplates overnight at 4°C. After a brief wash with PBST, the plates were blocked with 5% non-fat dry milk for 1 h at room temperature. Plasma samples diluted 1:20 in PBST were added to the plates and incubated for 2 h at room temperature. After washing with PBST, plates were incubated with 1:4000 rabbit anti-citrulline (Abcam) for 1 h at room temperature. The anti-citrulline antibody was labeled with alkaline phosphatase using the AP Labeling Kit (Roche) according to the manufacturer's instructions. The plates were then washed with PBST, and signals were detected as described above.

CEA levels were measured in all blood samples, AFP levels in liver cancer samples, CA199 levels in digestive tract tumor samples, CA125 levels in ovarian cancer samples, CA153 levels in breast cancer samples, CYFRA21-1 levels in lung cancer samples, and PSA levels in prostate cancer samples. All measurements were performed in a clinical laboratory cooperating with us.

ELISA data were collected from three independent tests. All statistical analyses were conducted using SPSS (version 11.0). The median and range of PADI4 and cAT levels are reported. The Mann-Whitney U-test statistically assessed differences between the groups, and the x² test was used to examine the association between expression of PADI4, cAT and several tumor markers. P values ≤ 0.05 were considered as statistically significant.

Total RNA was extracted using the TRIzol reagent (Invitrogen) from gastric adenocarcinomas (n = 5), lung adenocarcinomas (n = 5), hepatocellular carcinomas (n = 4), esophageal squamous cell cancers (n = 5), breast cancers (n = 5) and breast fibroadenomas (n = 5), as well as their corresponding healthy tissues. Concentrations of total RNA were determined with a spectrophotometer. Quantitative PCR analyses were performed using the iCycler Real-Time Detection System (Bio-Rad). Gene-specific primers were designed using the PADI4 mRNA sequence in Genbank (NM012387) and were as follows: forward 5'-ctgtggtgttccaagacagc-3' (nt position 871–890) and reverse 5'-gcttggatgtagccgatctc-3' (nt position 1083–1102). The TaqMan probe 5'-cccaacacccagcccccgca-3' (nt position 924–943) was labeled at the 5' end with the reporter dye FAM (6-carboxy-fluorescein) and at the 3' end with the quencher TAMRA (6-carboxy-teremethyl-rhodamine) (TaKaRa). cDNA was prepared with 1 ug of total RNA from each sample using random hexamers and reverse transcription with the PrimeScriptTM RT-PCR Kit (TaKaRa). A 232 bp DNA fragment was produced by 1st strand cDNA synthesis with the above primer set, cloned into the pGEM-T Easy Vector (Promega), and verified by sequence analysis. Purified recombinant plasmid was serially diluted to 1×10⁷, 1×10⁶, 1×10⁵, 1×10⁴, 1×10³ and 1×10² copies/ml. TaqMan real-time PCR was performed using the PrimeScriptTM RT-PCR Kit (TaKaRa) in 25 ul of reaction mixtures containing 12.5 ul of Premix Ex Taq, 0.5 ul of forward primer (10 uM), 0.5 ul of reverse primer (10 uM), 1 ul of TaqMan probe (3 uM), 2 ul of recombinant plasmid or cDNA and 8.5 ul of H₂O. The following optimized thermal cycling program was used: denaturation at 95°C for 3 min, followed by 50 cycles of 95°C for 30 s and 60°C for 30 s; fluorescence data were collected at the 60°C annealing and extension step. The PCR threshold cycle (Ct), which is defined as the fractional cycle number at which the fluorescence reaches 10 times the standard deviation of the baseline, was determined by the iCycler iQ software. Standard curve equations were calculated by regression analysis of the average Ct versus the log10 of the standard copy number. Copy numbers of PADI4 mRNA in clinical samples were calculated automatically by the data analysis software.