High STMN1 Expression Is Associated with Tumor Differentiation and Metastasis in Clinical Patients with Pancreatic Cancer

Materials and Methods

Clinical samples. We analyzed tumor specimens from 104 patients with PDAC who underwent excision surgery for a primary tumor between 1999 and 2012 at the Department of General Surgical Science of Gunma University School of Medicine and Gunma Prefecture Saiseikai-Maebashi Hospital, Japan. The patients included 56 men and 48 women. The age range was 36-87 years. The tumor stage was classified according to the seventh tumor-node-metastasis (TNM) classification of the Union for International Cancer Control (15) and the Sixth General Rules for the Study of Pancreatic Cancer of Japan Pancreas Society (16). Four patients had stage I, 84 had stage II, five had stage III and 11 had stage IV PDAC at the time of surgery. All patients signed written informed consent forms as required by our institutional guidelines.

Immunohistochemical staining. Sections (4-μm) of tumor and non-cancerous tissue were deparaffinized, rehydrated, and incubated with fresh 0.3% hydrogen peroxide in methanol for 30 min at room temperature to block endogenous peroxidase activity. The sections then were heated in boiled water and Immunosaver (Nishin EM, Tokyo, Japan) at 98°C for 45 min. Nonspecific binding sites were blocked by incubation with 10% rabbit pre-immune serum for 30 min. The sections were then incubated with primary monoclonal antibody to STMN1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at a dilution of 1:100 at 4°C overnight. The sections were washed in phosphate-buffered saline (PBS) and then incubated with biotinylated anti-mouse IgG for 60 min at room temperature (Nichirei, Tokyo, Japan). The chromogen 3,3’-diaminobenzidine tetrahydrochloride was applied as a 0.02% solution containing 0.005% H2O2 in 50 mM ammonium acetate-citrate acid buffer (pH 6.0). The sections were lightly counterstained with Mayer's haematoxylin and mounted. Negative controls were established by replacing the primary antibody with PBS. The immunohistochemical evaluation of STMN1 expression was confirmed independently by two observers. In positive cases, staining was primarily cytoplasmic. The intensity of STMN1 staining was scored as follows: 0: no staining, 1: weak-to-moderate staining, or 2: strong staining. A score of 0 for staining was considered to be low STMN1 expression (n=75), while scores of 1 and 2 were considered to be high STMN1 expression (n=29).

Cell culture. The human pancreatic cancer cell line Suit2 was used in this study. The cell line was obtained from the JCRB cell bank (Ibaragi, Osaka, Japan). The cells were cultured in RPMI 1640 medium (Wako, Osaka, Japan) supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin (Invitrogen, Carlsbad, CA, USA) in a humidified 5% CO₂ incubator at 37°C.

Establishment of a stable STMN1-transfected pancreatic cancer cell line. Human STMN1 complementary DNA (cDNA) was generated by reverse transcription-polymerase chain reaction (RT-PCR) and subcloned into PiggyBac Dual Promotor Vector PB513B-1 (System Biosciences, Palo Alto, CA, USA) using Ligation high Ver.2 (TOYOBO, Osaka, Japan) according to the manufacturer's protocol. Accurate in-frame insertion into the expression vector was confirmed by sequencing. Transfection into Suit2 cells was performed with PiggyBac Transposase Vector and SBI PureFection transfection reagents (System Biosciences) according to the manufacturer's protocol, and Suit2-STMN1 cells were established. A mock vector-transfected clone (Suit2-mock) was used as a control. Enhanced STMN1 expression was confirmed by RT-PCR and western blot analysis.

RNA extraction and quantitative real-time RT-PCR. Total RNA was extracted from Suit2 cells using the miRN-easy Kit (Qiagen, Hilden, Germany), and the quantity of isolated RNA was measured using an ND-10000 spectrophotometer (Nano-Drop Technologies, Wilmington, DE, USA). Quantitative real-time RT-PCR was performed using the GoTaq 1-Step RT-qPCR System (Promega, Madison, WI, USA) in a total volume of 20 μl. The program included four stages: reverse transcription at 37°C for 15 min; RT inactivation and hot-start activation at 95°C for 10 min; qPCR, of 40 cycles of 95°C for 10 s, 60°C for 30 s, and 72°C for 30 s; and dissociation at 60-95°C. The sequences of the primer pairs were as follows: STMN1 forward, 5’-CCGAGAAGAAGATCACCTTGAA-3’; STMN1 reverse, 5’-GACACGTCCTTCTTTTTGAAG C-3’; beta-actin forward, 5’-CCAACCGCGAGAAGATGA-3’; and beta-actin reverse, 5’-CCAGAGGCGTACAGGGATAG-3’.

Protein extraction and western blot analysis. Suit2-mock and Suit2-STMN1 cells were harvested at 80% confluence, and the total proteins were extracted using PRO-PREP Protein Extraction Solution Kit (iNtRON Biotechnology, Kyungki-Do, Republic of Korea). Total protein was electrophoresed on a 10% polyacrylamide gel, and then electroblotted at 300 mA for 90 min on a nitrocellulose membrane (Invitrogen). Western blotting was used to confirm the expression of STMN1 and heat-shock cognate protein 70 (HSC70) proteins: These proteins were detected using rabbit monoclonal antibody to STMN1 (1:1000; Cell Signaling Technology Inc., Danvers, MA, USA) and mouse monoclonal antibody to HSC70 (1:3000; Santa Cruz Biotechnology, Inc. Dallas, TX, USA). HSC70 expression was used as a loading control. The signals were detected using the ECL Western Blotting Detection System and an Image Quant LAS 4000 machine (GE Healthcare Life Sciences Inc., Chicago, IL, USA).

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Figure 1.

Immunohistochemical analysis of stathmin 1 (STMN1) expression in representative pancreatic ductal adenocarcinoma (PDAC) tissue samples. Examples of no STMN1 expression in non-cancerous pancreatic ducts (a), low STMN1 expression in a representative specimen of primary PDAC (b), high STMN1 expression in a representative primary PDAC specimen (c), high-power view of area shown in c (d) (Original magnification ×100).

Proliferation assay. Cell proliferation was measured with Cell Counting Kit-8 (Dojindo Laboratories, Kumamoto, Japan). Suit2-mock and Suit2-STMN1 cells were plated at 5,000 cells per 100 μl medium in 96-well plates. Evaluations were performed at 48 h. To determine cell viability, 10 μl of cell counting solution was added to each well and the plates further incubated at 37°C for 2 h. The absorbance of each well was then determined at 450 nm using a xMark Microplate Absorbance Spectrophotometer (Bio-Rad, Hercules, CA, USA).

Wound-healing assay. Suit2-mock and Suit2-STMN1 cells were plated in 6-well plates until confluence, and a uniform straight wound was produced in the monolayer in each well using a pipette tip. The wells were washed with PBS to remove all the cell debris, and the cells were cultured in 5% CO₂ at 37°C. The relative closure rate of the wound was quantitatively evaluated at 72 h using bright-field microscopy.

Statistical analysis. Differences between groups were estimated using Student's t-test, chi-square analysis and analysis of variance. A result was considered statistically significant when the relevant p-value was less than 0.05. All statistical analyses were performed usingJMP software (SAS Institute Inc., Cary, NC, USA).