During the period from January 2014 to December 2014, 78 consecutive patients were treated by complete surgical resection of lung adenocarcinoma at Kobe University Hospital, Kobe, Japan. Patients with a positive surgical margin were excluded from this study, and the remaining 74 patients were analyzed. The methods of data collection and analysis were approved by the institutional review board (permission number: 160117), and written informed consent was obtained from all patients.
Construction of the spiral array block and pathological studies
All surgical specimens were fixed with 10% formalin and embedded in paraffin. The paraffin-embedded block was sent to the Pathology Institute (Toyama, Japan) and processed into a spiral array block. The method of preparing the spiral array block is described in detail elsewhere . Briefly, 50- to 100-μm-thick slices of the sample block are cut and rolled up into cylindrical reels. These cylindrical reels are divided, and the reel containing the target site is embedded vertically in the recipient block. After that, the spiral array block is sliced and used for pathological examination. Serial 4-μm sections were stained with hematoxylin and eosin (Fig. 1A, B). All histologic specimens that had been initially evaluated by pathologists were reviewed, and the expression levels of SERPINE2 were assessed independently by two pathologists (N.J. and T.N.) who were blinded to the clinical data. The histological diagnoses were based on the 2015 WHO classification . Pathological stage was determined on the basis of the TNM classification of the International Union Against Cancer (UICC) .
Anti-human SERPINE2 antibody (Lot no. 00014298, Proteintech, Rosemont, IL, USA) was used as the primary antibody. The spiral array block was cut into 4 μm sections, which were mounted on silane-coated slides. The sections were deparaffinized in limonene (Nacalai, Kyoto, Japan) and dehydrated in a graded ethanol series. For antigen retrieval, the slides were heated for 20 min at 121 °C in 10 µM citrate buffer (pH 6.0), and endogenous peroxidase was blocked with 3% hydrogen peroxide in absolute methyl alcohol. The slides were then blocked in 2.5% horse serum for 1 h and incubated with the primary antibodies (1:200). After overnight incubation, the slides were then washed with phosphate–buffered saline and incubated with ImmPRESS Reagent (Vector laboratories, Burlingame, CA, USA). The reaction products were stained with ImmPACT DAB reagent (Vector laboratories), and the sections were counterstained with hematoxylin. A high SERPINE2 case was defined as one in which there was at least a 50% increase in positive cells compared to that in normal tissue (Fig. 1C-F).
The lung adenocarcinoma cell lines A549 and PC9 were obtained from the American Type Culture Collection (Manassas, VA, USA). All cells were cultured in RPMI 1640 medium (Sigma, St. Louis, MO, USA) supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin (Wako, Osaka, Japan) under 5% CO2 at 37 °C.
SERPINE2 knockdown by small interfering RNAs (siRNAs)
The SERPINE2 siRNAs (#1: s22188 and #2 s22189) and control siRNA (#14390843) were obtained from Thermo Fisher Scientific, MA, USA. Cells were plated in six-well plates at a density of 2 × 105 cells per well. The siRNAs or control siRNA duplexes were mixed with Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific) in Opti-MEM medium (Thermo Fisher Scientific) as described by the manufacturer’s protocol and added to the plated cells. The cells were used for assessments 24 h after the addition of fresh medium.
Quantitative reverse transcription-polymerase chain reaction (qRT-PCR)
Preparation of total cellular RNAs and qRT-PCR of the RNAs by using them as a template were performed as described previously . Relative mRNA levels were calculated with the ∆∆Ct method using GAPDH (glyceraldehyde-3-phosphate dehydrogenase) mRNA as an internal control. The primers used in this study were as follows: 5’-AATGAAACCAGGGATATGATTGAC-3’ and 5’-TTGCAAGATATGAGAAACATGGAG-3’ for SERPINE2 and 5’-GCACCGTCAAGGCTGAGAAC-3’ and 5’-ATGGTGGTGAAGACGCCAGT-3’ for GAPDH.
Cell proliferation assay
To determine the knockdown effects of SERPINE2 on cell growth, we used a cell proliferation assay (Cell Counting Kit-8 (CCK-8), Dojindo, Kumamoto, Japan) according to the manufacturer’s instructions. A549 and PC9 cells were plated at a concentration of 5 × 103 cells/well in 96-well culture plates and analyzed 72 h later.
Fas ligand stimulation
To investigate the involvement of SERPINE2 in the apoptosis pathway, lung cancer cell lines in which SERPINE2 was knocked down were stimulated with 200 ng/ml soluble Fas ligand (Wako, Osaka, Japan), and the temporal changes in apoptosis-related proteins were evaluated by western blotting.
Primary antibodies against the following proteins were purchased from Cell Signaling Technology (Denver, MA, USA): β-actin (#4967), Bax (#2772), Bcl-2 (#4223), cleaved caspase 7 (#8438), and cleaved caspase 9 (#9505). To detect SERPINE2, we used the same antibody used for the immunohistochemical analysis at a different dilution of 1:1000. Cells were lysed in Cell Lysis Buffer (Cell Signaling Technology), and total cellular proteins (20 µg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), which was followed by western blotting as described previously .
Evidence from TCGA database
To evaluate the relationship between SERPINE2 mRNA expression and the overall survival of lung adenocarcinoma patients, we utilized The Cancer Genome Atlas (TCGA) dataset through the Gene Expression Profiling Interactive Analysis (GEPIA) web server. We defined the patients in the 1st quartile in terms of SERPINE2 mRNA expression as the low SERPINE2 group and those in the 4th quartile as the high SERPINE2 group.
All statistical analyses were performed with EZR version 1.37 (Saitama Medical Center, Jichi Medical University; http://www.jichi.ac.jp/saitama-sct/SaitamaHP.files/statmed.html; Kanda, 2018), which is a graphical user interface for R (The R Foundation for Statistical Computing, Vienna, Austria, version 3.4.1) . Differences in patient characteristics between the two groups were tested for significance by the Pearson χ2 test or the Fisher exact test. For the univariate analysis, the cumulative survival was estimated by the Kaplan-Meier method, and differences in variables were calculated by the log-rank test. A multivariate regression analysis was conducted according to the Cox proportional hazards model. All reported P values are 2-sided, and a P value less than 0.05 was considered significant.