Chloride Intracellular Channel 1 Expression Is Associated With Poor Prognosis of Lung Adenocarcinoma

Patients and Methods

Patients. We analyzed 74 consecutive patients who underwent complete surgical resection of adenocarcinoma at Kobe University Hospital between January 2014 and December 2014. Pathological staging was determined based on the seventh edition of the TNM classification for lung cancer (8). 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.

Spiral array. The method of constructing the spiral array block has been described in detail previously (9).

Briefly, the tissue sample block was sent to Pathology Institute Corporation (Toyama, Japan). Then, 50- to 100-μm-thick slices of the sample block were cut and rolled up into cylindrical reels. One of the reels was embedded vertically in the recipient block to create a spiral array block. Finally, 4.0-μm sections were created for histopathological analysis.

Immunohistochemistry. Immunohistochemistry was performed using a standard procedure previously reported (10). Briefly, tissue sections from spiral array blocks were deparaffinized in limonene and dehydrated in a graded alcohol series. For activation of antigen by heating, the slides were heated for 20 min at 121°C in 10 μM citrate buffer (pH 6.0) in an autoclave. Endogenous peroxidase was blocked with 3% hydrogen peroxide in absolute methyl alcohol. Then, the slides were blocked in 2.5% horse serum for 60 min at room temperature. Next, a primary antibody against CLIC1 (Proteintech, IL, USA) was applied to slides at 4°C overnight (1:200). The slides were incubated with ImmPRESS Reagent (Vector Laboratories, CA, USA) for 30 min. Finally, DAB staining was performed using ImmPACT DAB reagent (Vector Laboratories). High expression was defined as a case in which the number of positive cells increased by 50% or more compared to those of normal tissue (Figure 1).

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Figure 1.

The upper images show typical negative staining for CLIC1 of the spiral array specimens (A, B, C). The lower images show typical positive staining for CLIC1 (D, E, F).

Cell culture. The lung adenocarcinoma cell lines A549 and PC9, human large cell lung cancer cell line H460, human lung squamous cell carcinoma cell line H520, and human small cell lung cancer cell lines DMS53 and DMS114 were purchased from ATCC (Manassas, VA, USA). The lung adenocarcinoma cell line II18 was purchased from RIKEN Cell Bank (Ibaraki, Japan). These cells were cultured at 37°C with 5% CO-1640 medium (Wako, Osaka, Japan) containing 10% fetal bovine serum and 1% penicillin–streptomycin (Nacalai Tesque, Kyoto, Japan).

CLIC1 knockdown by small interfering RNAs. CLIC1 siRNAs (#1: s635 and #2: s636) and negative control siRNA (#4390843) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Knockdown was performed according to the manufacturers’ protocol. Briefly, cultured cells were confirmed to be 60-80% confluent in 6-well plates. The siRNA or control siRNA was mixed with Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific) in Opti-MEM medium (Thermo Fisher Scientific), and the mixture was then added to the cells. The cells were collected and analyzed 24 or 48 h after knockdown.

Reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR. Total cellular RNA extraction and RT-PCR were performed as previously reported (11). Extraction of total cellular RNA was performed using Sepasol-RNA I Super G (Nacalai Tesque) according to the manufacturer’s protocol. Complementary DNA was generated using SuperScript III First-Strand Synthesis SuperMix for real-time PCR (Thermo Fisher Scientific). qRT-PCR was performed using a Thermal Cycler Dice Real Time System II (TaKaRa, Shiga, Japan). Relative mRNA levels were calculated with the ΔΔCt method using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA as an internal control. The primers used in this study were as follows: 5’-ACCGCAGGTCGAATTGTTC-3’ and 5’-ACGGTGGTAACATTGAAGGTG-3’ for CLIC1 and 5’-GCACC GTCAAGGCTGAGAAC-3’ and 5’-ATGGTGGTGAAGACGCC ATG-3’ for GAPDH.

Cell proliferation assay. A549 and PC9 cells in the logarithmic growth phase were seeded in 96-well plates at a density of 5,000 cells/well and precultured in a 5% CO₂ incubator for 24 h. CLIC1 siRNA and negative control siRNA were transfected into these cells, and the cells were cultured at 37°C in a 5% CO₂ incubator. Then, a total of 10 μl of Cell Counting Kit-8 (CCK-8) solution (Dojindo, Kumamoto, Japan) was added to each well, and the cells were incubated for another 120 min. Absorbance was measured at 450 nm using a spectrophotometer (Bio-Rad, iMark microplate reader, CA, USA).

Scratch assay. A scratch assay was performed as described previously (12). A549 and PC9 cells were seeded in 6-well plates. When the cells grew to 100% confluency after knockdown of CLIC1, a monolayer was scratched using a p200 pipette. Images were taken under a BZ-X700 fluorescence microscope (Keyence, Osaka, Japan) at different times according to the growth speed of the cells. The wound width was analyzed by ImageJ (13).

Western blotting. A protocol for western blotting has been described previously (14). The amount of protein used in this study was standardized at 20 μg. Primary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA): β-actin (#4967), p38 MAPK (#9212), p-p38 MAPK (#9215), cleaved caspase 3 (#9661), BAX (#2772) and Bcl2 (#4223). The primary antibody for CLIC1 was the same antibody used for immunohistochemical staining.

Statistical analysis. Fisher’s exact test or Pearson χ² tests were used for between-group comparisons of the categorical variables. For the univariate analysis, cumulative survival was estimated by the Kaplan–Meier method, and differences were assessed by the log-rank test. A multivariate regression analysis was conducted according to the Cox proportional hazard model. All p-values reported are 2-sided, and p-values less than 0.05 were considered significant. All statistical analyses were performed with EZR (Saitama Medical Center, Jichi Medical University), which is a graphical user interface for the R software program (The R Foundation for Statistical Computing, version 2.13.0) (15). More precisely, it is a modified version of the R commander (version 1.27) that includes statistical functions that are frequently used in biostatistics.