Oncology Research, Vol. 21, pp. 271–279 Printed in the USA. All rights reserved. Copyright Ó 2014 Cognizant Comm. Corp.

0965-0407/14 $90.00 + .00 DOI: http://dx.doi.org/10.3727/096504014X13946737557031 E-ISSN 1555-3906 www.cognizantcommunication.com

SiRNA-Mediated Flotillin-2 (Flot2) Downregulation Inhibits Cell Proliferation, Migration, and Invasion in Gastric Carcinoma Cells Ke Cao,* Dingfang Xie,* Peiguo Cao,* Qiong Zou,† Can Lu,* Sheng Xiao,† Jianda Zhou,‡ and Xiaowei Peng§ *Department of Oncology, The Third Xiangya Hospital, Central South University, Changsha, Hunan, China †Department of Pathology, The Third Xiangya Hospital, Central South University, Changsha, Hunan, China ‡Department of Plastic Surgery, The Third Xiangya Hospital, Central South University, Changsha, Hunan, China §Department of Head and Neck Surgery, The Affiliated Tumor Hospital of Xiangya Medical School, Central South University, Changsha, Hunan, China

The flotillin (Flot) protein family has been demonstrated to be involved in the development and progression of various cancers. However, the role of Flot2 in gastric carcinomas remains unknown. The present study aimed to investigate the clinical significance and the role of Flot2 in gastric carcinomas. Data of tissue microarray including 90 cases of gastric carcinoma samples and their matched adjacent tissues showed that, among 90 cases of adjacent tissues, 65 cases showed no Flot2 expression, and 25 cases showed low expression of Flot2, and its positive expression rate was only 38.5% (25/90); however, among 90 cases of gastric carcinomas, 6 cases showed no Flot2 expression, 26 cases showed low Flot2 expression, 28 cases showed moderate expression of Flot2, and 30 cases showed high expression of Flot2, and its positive expression rate was 93.3% (84/90). Moreover, the Flot2 expression was significantly associated with the histological grade, depth of invasion, lymph node metastasis, and TNM stage. Furthermore, data of survival analysis suggested that Flot2 protein expression was an independent prognostic factor of poor survival. After that, Flot2-specific siRNA was used to decrease the Flot2 expression in gastric cancer AGS and SGC7901 cells. Forced downregulation of Flot2 remarkably inhibited cellular proliferation, migration, and invasion in gastric carcinoma cells. In conclusion, the present study suggests that the Flot2 protein expression is significantly correlated with cancer progression and poor prognosis in gastric carcinomas, probably due to its role in the regulation of cell proliferation, migration, and invasion in gastric carcinoma cells. Key words: Flotillin2 (Flot2); Gastric carcinoma; Prognosis; Proliferation; Migration; Invasion

INTRODUCTION Flotillin (Flot) is a protein family on microdomain lipid rafts, which have been reported to play a role in various biological processes, including cell survival, proliferation, adhesion, apoptosis, and motility, mainly due to its implication in vesicular invaginations of the plasma membrane, signal transduction pathways, organization of the cytoskeleton, protein sorting during both exocytosis and endocytosis, as well as synaptic transmission (1–3). Among flotillin members, Flot2 has been demonstrated to be involved in several cancers, such as melanoma, esophageal squamous cell carcinoma, and colorectal cancer, as well as gastric cancer (4–8). Further studies have revealed that Flot2 is an important tumor regulator mainly via interacting with other molecules including kinases, receptors, and adhesion molecules, as well as G proteins (4). However, the role of Flot2 in gastric carcinomas remains unknown.

The present study first evaluated the clinicopathologic significance of the Flot2 protein expression in gastric carcinoma samples, as well as their matched adjacent tissues by performing tissue microarray assay as well as statistical analysis. We further determined whether the Flot2 protein expression was an independently prognostic factor for the 5-year survival of patients with gastric cancer. Moreover, we preliminarily investigated the role of Flot2 in cell proliferation, migration, and invasion in gastric carcinoma cells. In sum, the present study suggests that Flot2 may become a potential candidate for the development of prognostic and therapeutic strategies for gastric carcinomas. MATERIALS AND METHODS Tissue Microarray Assay Tissue microarray (HStm-Ade180Sur-01) was purchased from Core Ultra Biological Technology Co., Ltd. (Shanghai,

Address correspondence to Peiguo Cao (Professor), Department of Oncology, The Third Xiangya Hospital of Central South University, Tongzipo Road, Changsha, Hunan 410013, P.R. China. Tel: +86-731-8861-8804; Fax: +86-731-8529-5260; E-mail: [email protected]

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272 cao ET AL.

China). This microarray included 90 cases of gastric carcinomas as well as their matched adjacent tissues, which were normal gastric mucosa 2 cm away from cancer tissues. The age was from 34 to 83 years old, with a median age of 65.5 years old. Sixty-seven cases were male, and 23 cases were female. Pathological grading was five cases of grade I, 31 cases of grade II, and 54 cases of grade III. According to the seventh edition of esophageal cancer of the American Joint Committee on Cancer staging using UICC, TNM clinical staging was as follows: 6 cases of stage I, 30 cases of stage II, 51 cases of stage III, and 3 cases of stage IV. The pathological diagnosis of each patient was adenocarcinoma conformed by H&E staining. All patients underwent surgery and diagnosis from May 2007 to February 2008. No chemotherapy or other anticancer therapy was performed before surgery. All patients had complete follow-up data. The follow-up deadline was August 2012. Since 59 cases died during follow-up, the shortest follow-up time was 1 month, and the longest follow-up time was 63 months, with a median survival time of 26 months. Tissue microarray was examined and scored independently by two experienced pathologists. Flot2 expression was evaluated according to the ratio of positive cells per specimen and staining intensity. The mean percentage of positive tumor cells was determined in at least five random fields at 400× magnification in each section. The intensity of the Flot2 immunoreaction was scored as follows: 1+, low; 2+, moderate; and 3+, high. The percentage of positive tumor cells and the staining intensity then were multiplied to produce an immunohistochemical staining score.

Transfection Cells were cultured to 70%–80% confluence and then were resuspended in serum-free H-DMEM at a concentration of 100,000 cells/ml. Six-well plates were used to inoculate with 2 ml suspension for each well. Flot2specific siRNA or nonspecific siRNA of 50 pmol was diluted with 0.25 ml serum-free H-DMEM; 50 μl of lipo2000 transfection reagent was diluted with 2.5 ml serum-free H-DMEM. Then, the diluted lipo2000 transfection reagent was added into the dilution above, mixed gently, and incubated for 20 min at room temperature. The cell suspensions were washed with serum-free H-DMEM for two times and then added with the mixture of lipo2000 and siRNA above, and then incubated at 37°C, 5% CO2 for 6 h. After that, the medium in each well was replaced by the normal serum-containing medium and cultured for 24 h before the following experiments. Western Blot Cold RIPA lysis buffer was used to solubilize cells. Protein assay reagents (Bio-Rad Laboratories, Hercules, CA, USA) were used to determine the protein concentration. Protein was then separated with 12% SDS-PAGE and transferred to a PVDF membrane, which was then blocked in 5% nonfat dried milk in PBS at room temperature for 4 h. The membrane was then incubated with mouse antiFlot2 monoclonal antibody (1:200, Abcam, Cambridge, UK) or mouse anti-b-actin monoclonal antibody (1:400, Abcam, Cambridge, UK) at room temperature for 3 h. The membrane was then incubated with rabbit anti-mouse secondary antibody (1:40,000, Abcam, Cambridge, UK) for 40 min. Enhanced chemiluminescence reagent detected the signal on the membrane. Data were analyzed by densitometry using Image-Pro plus software 6.0 and normalized to b-actin expression.

Materials and Agents High-glucose Dulbecco’s modified Eagle’s medium (H-DMEM), TRIZOL, MTT, and RT-PCR Kit were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Fetal bovine serum (FBS) was purchased from Tianhang Biology (Hangzhou, Zhejiang, China). MTT was purchased from Biosharp (Hefei, Anhui, China). All siRNAs were designed and produced by GenePharma (Shanghai, China). SYBR Green qRCR Mix was purchased from TOYOBO (Osaka, Japan). Mouse anti-Flot2 monoclonal antibody and rabbit anti-mouse secondary antibody were purchased from Millipore (Boston, MA, USA). The Transwell chamber was obtained from Corning Incorporated (Corning, NY, USA).

Cell Proliferation Assay For all groups, 10,000 cells per well were plated in a 96-well plate. After treatment, the plates were incubated for 0, 24, 48, or 72 h at 37°C, 5% CO2. To assess cell proliferation, MTT assay was performed according to the manufacturer’s manual; 50 μl 5 mg/ml MTT reagent in PBS was added to each well and incubated for 4 h at 3°C, 5% CO2. Then, the supernatant was removed, and 150 μl of DMSO was added. The absorbance was detected at 570 nm with a Microplate Reader (Bio-Rad, USA). Each assay was performed in triplicate wells and repeated three times.

Cell Culture Human gastric carcinomas AGS and SGC7901 cells were purchased from the Cell Biology Laboratory of Xiangya Medical College, Central South University. Cells were cultured in DMEM medium containing 10% FBS at 37°C with 5% CO2.

Scratch Assay Cells in each group were collected and resuspended in H-DMEM containing 10% FBS. Then, each well of a 24-well plate was seeded with 50,000 cells. When these cells were cultured at 37°C, 5% CO2 into about 100% confluence, we scratched the bottom with the head of a

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Flot2 DOWNREGULATION INHIBITS GASTRIC CARCINOMA

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10-μl tip and washed these cells with serum-free medium. Then, serum-free medium was used to culture these cells at 37°C, 5% CO2 for 24 h. After that, we replaced it with H-DMEM medium containing 10% FBS and continued to culture them at 37°C, 5% CO2. Three days later, we photographed these cells of each group.

cotton bud was used to remove the cells that were not through the polycarbonate membrane. Then, the cells that transmembraned through the polycarbonate membrane and adhered to the bottom of it were stained with trypan blue for 15 min and then were photographed and counted. After that, each well was added with 500 μl 10% ethanol to dissolve the dye on the polycarbonate membrane. Then, cells were transferred to a 96-well plate to measure the absorbance at 570 nm using a Microplate Reader. Each assay was performed in triplicate wells.

Cell Invasion Assay The cell invasion assays were performed in a 24-well Transwell chamber, which was precoated with 100 μg Matrigel. Cells in each group were collected and resuspended in serum-free H-DMEM at a concentration of 10,000 cells/ml, respectively. Then, 0.2-ml cell suspensions were added into the upper chamber, and the bottom chamber was filled with 0.5 ml H-DMEM containing 10% FBS. After incubation for 24 h at 37°C, 5% CO2,

Statistical Analysis All data were expressed as mean ± SD from at least three separate experiments. SPSS 17.0 software was used for statistical analysis. The association between Flot2 protein expression and clinicopathologic features in patients

Figure 1.  The expression of Flot2 in normal gastric mucosa, high differentiated gastric adenocarcinoma, and low differentiated gastric adenocarcinoma. Flot2 expression in normal gastric mucosa or gastric adenocarcinoma tissues is shown in six representative results of immunohistochemical staining in the tissue microarray. (A) Normal gastric mucosa (200×). (B) Normal gastric mucosa (400×). (C) High differentiated gastric adenocarcinoma (200×). (D) High differentiated gastric adenocarcinoma (400×). (E) Low differentiated gastric adenocarcinoma (200×). (F) Low differentiated gastric adenocarcinoma (400×).

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274 cao ET AL.

with gastric cancer was analyzed using chi-square test and Spearman rank correlation analysis. Survival curves were determined using Kaplan–Meier curves and a log-rank test. Multivariate prognostic factors were examined using Cox’s proportional hazards model. The statistical correlation of data between groups in cell experiments was analyzed by one-way analysis of variance (ANOVA). A value of p