TFF1 inhibits proliferation and induces apoptosis of gastric cancer cells in vitro

Trefoil Factor Family (TFF) plays an essential role in the intestinal epithelial restitution, but the relationship between TFF and gastric cancer (GC) is still unclear. Th e present study aimed to determine the role of TFF in repairing gastric mucosa and in the pathogenesis of GC. Th e TFF expression in diff erent gastric mucosas was measured with immunohistochemistry. Th en, siRNA targeting TFF or plasmids expressing TFF gene were transfected into BGC cells, SGC cells and GES- cells. Th e cell proliferation was detected with MTT assay and apoptosis and cell cycle measured by fl ow cytometry. From normal gastric mucosa to mucosa with dysplasia and to gastric cancer, the TFF expression had a decreasing trend. Down-regulation of TFF expression signifi cantly reduced the apoptosis of three cell lines and markedly facilitated their proliferation but had no signifi cant eff ect on cell cycle. Over-expression of TFF could promote apoptosis of three cell lines and inhibit proliferation but had no pronounced eff ect on cell cycle. TFF can inhibit proliferation and induce apoptosis of GC cells in vitro. ©  Association of Basic Medical Sciences of FBIH. All rights reserved


INTRODUCTION
Although the incidence of gastric cancer (GC) has declined over the past  years worldwide, especially in western countries, it remains the second leading cause of cancer-related death and accounts for . of cancer deaths globally []. About  GC cases and  cancer deaths are estimated to have occurred in  worldwide []. More than one-half of GC patients have lymph node metastases when they are initially diagnosed or operated, which usually results in poor prognosis [-]. Therefore, it is important to investigate the pathogenesis of GC, fi nd eff ective measures to prevent and treat GC. Th e trefoil factor family (TFF), which comprises gastric peptides pS/TFF and spasmolytic peptide (SP)/TFF and intestinal trefoil factor (ITF)/TFF, plays an essential role in the intestinal epithelial restitution []. Th ey are small (~ kDa) protease-resistant proteins and are abundantly secreted onto the mucosal surface by mucus-secreting cells in the gastroin-testinal tract. Th e TFFs share an absolutely conserved distinct motif of six cysteine residues that defi ne a so-called "trefoil" domain, which is also known as a "P" domain []. At certain physiological conditions, in the presence of a tissue-specifi c distribution, TFF plays an important role in mucosal protection and wound healing. But in malignant tissues, TFF is highly expressed and correlated strongly with the genesis, metastasis and invasion of tumor cells. Th ese indicate that TFF may be a common mediator of oncogenic responses to diff erent stimuli. Th e biological functions of TFF involve complex regulatory processes. TFF was fi rst discovered in breast cancer cell line MCF- in  [-]. In normal tissues, the main site of expression of TFF is the mucosal epithelial cells of gastric body and antrum in a site-specifi c fashion. However, under pathological conditions, such as ulceration, the TFF-expression of site-specifi c fashion is absent, TFF can be identifi ed in any damaged mucosas, and its expression is up-regulated to participate in gastrointestinal epithelial reconstruction and repair process []. However, there is lack of TFF expression in human GC. To determine the function of TFF, the mouse TFF gene was inactivated. Th e antral and pyloric gastric mucosa of mpS-null mice was dysfunctional and exhibited severe hyperplasia and dysplasia. All homozygous mutant mice developed antropyloric adenoma, and  developed multifocal intraepithelial or intramucosal carcinomas. Th ese results indicate that TFF is essential for the nor-mal diff erentiation of antral and pyloric gastric mucosa and may function as a gastric-specifi c tumor suppressor gene []. Th e aim of our study is to determine the role of TFF in repairing gastric mucosa and in the pathogenesis of GC. To this end, the TFF expression was detected in different gastric mucosas. Then, siRNA targeting TFF and plasmids expressing TFF were transfected into normal gastric mucosal epithelial cells (GES- cells []), highly malignant GC cell line (BGC cells) and moderately malignant GC cell line (SGC cells), respectively, to investigate the effect of TFF on the biological behaviors of gastric mucosal epithelial cells, which provides evidence for further investigation and application of TFF target therapy.

MATERIALS AND METHODS
Sample collection and immunohistochemistry GC tissues and adjacent normal and atypical hyperplasia gastric mucosas were obtained from  patients undergoing gastroscopy at Tongji Hospital of Tongji University. Th e characteristics of these patients are shown in Table . All antibodies used for immunohistochemistry were purchased from Zhongshan Goldenbridge Biotechnology CO., LTD (Beijing, China). All other chemicals and reagents were commercially available and had the highest purity. Tissues were fi xed in  formaldehyde in phosphate buff ered saline (PBS), embedded in paraffi n, and cut into -μm sections. Sections were heated at  o C overnight, deparaffi nzed with xylene twice, and rinsed in a decreasing ethanol series (-) for  min/solution. Samples were treated with  HO for  min to inactivate endogenous peroxidase. Antigen retrieval was done with . M Na-citrate buffer (pH .) in a microwave oven for  min. Sections were incubated at  o C overnight in moist chambers with primary antibody (:) and then with biotinylated secondary antibody (Zhongshan Goldenbridge Biotechnology CO., LTD., Beijing, China, SP-Kit) for  min at room temperature, followed by incubation with streptavidin peroxidase. Visualization was done with diaminobenziding tetrachloride and counterstaining was performed with haematoxylin. In negative controls, the primary antibody was replaced with PBS. Sections were observed under a light microscope and positive cells had brown granules in cytoplasm. Five fi elds were randomly selected from each section at high magnification, and  cells were counted in each field, followed by calculation of percentage of positive cells. Sections with positive cells of > was regarded as positive.

Cell culture
Two gastric adenocarcinoma cell lines (BGC cells and SGC cells) (Cell bank of Chinese Academic of Scienc-es), as well as normal gastric epithelial cell line (GES- cells) (Tumor Institute of Beijing Medical University, China) were maintained in RPMI  medium (Gibco BRL, USA) supplemented with  fetal bovine serum (FBS; Hangzhou Sijiqing, China) and  μg/ml streptomycin and penicillin G (Amresco, USA) at  o C under  humidifi ed CO. Passaging was performed every  days by trypsinization (Sigma, USA). Synthesis of TFF-siRNA and Cell Transfection mRNA sequence of TFF was obtained from GeneBank. With Invitrogen's online design software BLOCK-iTTM RNAi Designer, three sites (stealth , stealth , stealth ), and they were selected and designed to be three sets of targeting stealth siRNA sequence. Th e selection and design were based on three principles: avoiding the ' and ' end non-coding region, selecting the sequences with G/C ratio between  and  and using BLAST to exclude other coding sequences. Stealth siRNA sequences are shown in Table . When the cell confl uence reached about ~, transfection of Stealth siRNAs and stealth RNA negative control was performed with LipofectamineTM according to manufacturer's instructions (Invitrogen, USA). Cells transfected with Stealth siRNAs was defined as Stealthgroup, those transfect with Stealth negative control as negative control group, and those without transfection as blank control group. Detection of TFF mRNA expression by RT-PCR Total RNA was extracted with Trizol reagent (Invitrogen, USA). About  μg of RNA were used for reverse transcription with random primers to synthesize fi rst strand cDNA, followed by conventional PCR amplification with  μl of cDNA as template. Th e forward primers, reverse primers and anticipated size of products and GAPDH are shown in Table  . Th e volume of reaction system was  μl, and reaction conditions were as follows: denaturation at  o C for  min; an-    nealing at  o C for  min; extension at  o C for  min. Th e products were then subjected to agarose gel electrophoresis, and images were captured to analyze the quality of RNA.

Determination of cell proliferation with MTT assay
Cell viability was determined by MTT assay. Different cell lines were seeded at   /ml into -well plates, and then divided into blank control group, blank transfection group, stealth_ group, stealth_ group and stealth_ group. At  h after transfection, the cell proliferation was determined by MTT assay every other  h for  days. In brief, at designed time points, MTT was added into each well at a final concentration of . mg/ ml followed by incubation for  h at  o C. Th e formazan was dissolved by addition of dimethylsulfoxide (DMSO) and absorbance (A) was measured with microplate reader (Bio-Rad, USA) at  nm. Th e inhibition rate (IR) was calculated formulas follow: IR = (-A experiment /A control )×.

Detection of cell cycle by fl ow cytometry
At  h after transfection, cells (×  cells) were harvested and washed twice in cold PBS. Cells were fi xed in  ethanol and washed in cold PBS. Th en, the cells were suspended in  ml of propidium iodide (PI, Sigma, USA) solution containing  μg/ml PI,  μg/ml RNAase (Sigma, USA), . (w/v) sodium citrate and . (v/v) Triton X. Cells were incubated at room temperature in dark for at least  min, and analyzed by flow cytometry (Beckman, USA).

Detection of cell apoptosis by fl ow cytometry
At  h after transfection, ×  cells were harvested. According to the instructions in Annexin V-FITC kit (Nanjing Keygen Biotech. Co. Ltd., China), cells were suspended in  μl of binding buffer and  μl of Annexin V-FITC followed by addition of  μl of PI and subsequent incubation for  min at room temperature in dark. Apoptosis was detected by flow cytometry.

Construction of plasmid TFF-pcDNA. and cell transfection
Human plasmid TFF-pcDNA. was constructed and identified by Shanghai Shuiyuan Biotechnology Company. Cells were divided into TFF-pcDNA. transfection group, pcDNA. negative control group and blank control group. Cells were seeded into -well plates. When cell confluence reached about , transfection was performed with LipofectamineTM according to manufacturer's instructions (Invitrogen, USA).

Detection of TFF protein expression by western blot
Total protein was extracted and  μg of proteins were subjected to polyacrylamide gel electrophoresis. Th en, the proteins were transferred onto PVDF membrane, which were blocked for  h at  o C in  non-fat milk and then incubated with mouse anti-human TFF or GAPDH monoclonal antibody (Santa Cruz, USA; :) for . h at room temperature. After washing in PBST thrice ( min for each), the membrane was incubated with HRPconjugated goat anti-mouse secondary antibody (Santa Cruz, USA; :) at room temperature for . h. Following rinsing in PBS, visualization was done and images were captured with a Touching gel imaging system. In negative control group, primary antibody was replaced with PBS.

Determination of cell proliferation with MTT assay
Different cell lines were inoculated at   /ml into -well plates, and then divided into blank control group, TFF-pcDNA.-transfection group and pcDNA. transfection group. At  h after transfection, cell proliferation was measured with MTT assay every other  h for  days. The procedures of MTT assay were abovementioned.

Detection of cell cycle by fl ow cytometry
At  h after transfection, cells (×  cells) were harvested and processed with above procedures. Cell cycle was measured by fl ow cytometry.

Detection of cell apoptosis by fl ow cytometry
At  h after transfection, ×  cells were harvested and processed with above procedures. Cell apoptosis was detected by fl ow cytometry.
Statistical analysis SPSS version .. statistical software was employed for statistical analysis and data were expressed as means ± standard deviation (X ± s). Independent sample t-test was used to compare data between two groups and one-way ANOVA to compare date among multiple groups. If there were significant differences, a further least significant difference method would be used for pairwise comparison. A value of p <. was considered statistically significant.

RESULTS
TFF Expressionin diff erent mucosal tissues TFF were mainly expressed in the cytoplasm of gastric mucosal cells. Perinuclear accumulation was the most obvious and positive cells were stained brown. Th e closer to the cell membrane is, the deeper the color is. Th e positive expression rate of TFF in normal gastric mucosa was  (/). In mucosas with dysplasia, the TFF expression was slightly reduced and the positive expression rate was . (/). From normal gastric mucosa, dysplasic mucosa to GC, the TFF expression had a gradually decreasing trend, and significant difference in TFF expression was noted among groups (Table , Figure ). Th e positive expression rate of TFF was . in males (/) and . in females (/) showing no significant difference. The positive expression rate of TFF was . in patients aged ≥  years (/) and . in those aged < years (/) showing no marked difference. These results suggest that TFF expression was independent of both age and gender (Table ).

Stealth siRNA inhibited mRNA expression of TFF
Results from RT-PCT showed stealth siRNA significantly inhibited TFF expression in a time dependent manner. At  h after transfection, the inhibition of TFF expression was present, and then reached a maximal level at  h after transfection but became to reduce at  h after transfection. Different stealth siRNAs inhibited the TFF expression when compared with control group and blank transfection group. Th e inhibitory eff ect of stealth_ was the most obvious and, at  h after transfection, the inhibition rate of TFF expression in GES- cells, BGC cells and SGC cells was ., . and ., respectively. The inhibitory effect was comparable among  different cell lines. Although TFF expression in negative control group was slightly lower than that in control group, there was no significant difference (p>.) ( Figure ).

Down-regulation of TFF reduced apoptosis rate and promoted cell proliferation but had no eff ect on cell cycle
MTT assay showed that the proliferation of three cell lines undergoing transfection with stealth siRNAs increased significantly at  h and  h after transfection (p<.), and the increase in steath_ group was the most obvious and reached a peak level at  h after transfection. Th e proliferation remained comparable among cells at  h,  h and  h after transfection (p>.) (Table , Figure ). Th e apoptosis rate of three cell lines was signifi cantly reduced at  h after TFF stealth siRNA transfection when compared with control group (p<.). Th e reduction of apoptosis rate in stealth_ group was most obvious and the apoptosis rate in BGC cells, SGC cells and GES- cells was reduced by ., . and ., respectively. However, there was no *p < 0.05 vs normal gastric mucosa   Table ).

TFF protein expression after transfection with TFF-pcD-NA.
When compared with TFF expression before transfection, the TFF protein expression in GES- cells, BGC cells and SGC cells was markedly increases (p<.) in a time dependent manner. Increase of TFF expression was noted as early as  h after transfection, but the TFF expression was similar to that in control group (p>.). TFF expression reached a peak level at  h after transfection but began to reduce at  h after transfection ( Figure )

Over-expression of TFF promoted apoptosis, inhibited cell proliferation but had no eff ect on cell cycle
MTT assay showed that cell proliferation reduced after TFF-pcDNA. transfection,. Th e IR was the most obvious at  h after transfection (p<.). Th e IRs of GES- cells, BGC     Figure ). The apoptosis rate of TFF-pcDNA. transfected cells markedly increased at  h after transfection when compared with control group (p<.). The apoptosis rate increased from . ± . to . ± . in GES- cells, from . ± . to . ± . in BGC cells, and from . ± . to . ± . in SGC cells (Table ). After TFF-pcDNA. transfection, the proportion of cells in G and G/M phase markedly decreased, while that in S-phase significantly increased, indicating that cells were arrested in S phase. The cell cycle distribution was markedly diff erent from that in control group (p<.) ( Table ).

DISCUSSION
Under physioligical conditions, human TFF is mainly expressed in epithelial cells of gastrointestinal tract. TFF expression has regional and cellular selectivity, and, in normal tissues, TFF is expressed in the gastric antrum, the crypt of gastric body and mucosa. Low TFF expression is also noted in the small intestine, colon and breast epiderm. However, under pathological conditions, the specifi city in TFF expression is absent. When the gastrointestinal mucosa is injured, TFF may be expressed in the injured mucosa of gastrointestinal tract, and its expression is rapidly up-regulated to participate in the reconstruction and repair process of epithelial cells in gastrointestinal tract. Our study found that, in normal gastric mucosa, the TFF expression was mainly found in gastric epithelial cells, gastric pits and glands, which was consistent with previous study. In addition, TFF is now considered as a tumor suppressor, and may be a stomach-specifi c suppressor factor. In humans, about  of GC patients show loss of human pS expression. It is generally believed that GC is preceded by a precancerous progress with the following well-recognized steps: normal gastric mucosa, chronic active inflammation, multifocal atrophy (gland loss), intestinal metaplasia, complete type-intestinal metaplasia, incomplete type-dysplasia [, ]. The accumulation of many genetic and molecular changes is closely  related to the evolution []. Taupin et al. [] investigated the expressions of pS and ITF in diff erent gastric mucosal injury models. Results showed that, in the gastric mucosa metaplasia-dysplasia-gastric cancer process, the TFF expression was gradually reduced, and TFF expression was lost earlier than the diff erentiation of gastric epithelial metaplasia, suggesting that loss of TFF expression is an early event in the occurrence of GC. Our results confi rmed that TFF expression had a decreasing trend from normal gastric mucosa, gastric dysplasia to GC, consistent with previous findings.
In recent years, studies have shown that TFF is relevant with cell proliferation and apoptosis to some extents. Rodrigues et al. [] found that, in colon cancer cells undergoing transfection of TFF, cell dispersion was promoted possibly through cyclooxygenase (COX) and thromboxane A (TXA-) receptor dependent mechanism, enhancing the ability of cell infi ltration. Another study [] indicated that TFF played a dual role on the gastrointestinal cells: on one hand, TFF can block the transition from G phase to S phase, which interrupts the gastrointestinal cell diff erentiation and reduces cell proliferation; on the other hand, TFF prevents chemical factor-induced apoptosis. Th ese reveal TFF is a regulatory factor of gastrointestinal cell diff erentiation. To reduce cell proliferation and induce diff erentiation are functional characteristics of tumor suppressors. Apoptosis refers to the programmed cell death in the growth, development and diff erentiation of cells and pathological environments. It is a type of cell death diff erent from necrosis. Recent studies have found that apoptosis plays a unique role in the renewing of gastrointestinal mucosa and in the pathological processes of some digestive diseases. TFF exerts its anti-tumor eff ect through regulating the balance between cell proliferation and apoptosis. Taupin et al. [] investigated the infl uence of TFF and its mutants on apoptosis and results showed that TFF trefoil domain mutant and alteration could promote cell migration, and increase the resistance to death during the process of migration. Th e mutations of trefoil domain were also detectable in TFF, a protein with similar structure to TFF, and this region is critical for the anti-tumor eff ect of TFF []. In our study, the TFF mRNA expression was inhibited by Stealth siRNA transfection, and the TFF was over-expressed followed transfection of plasmids expressing TFF gene. After inhibition of TFF expression by siRNA targeting TFF, the proliferation rate of human GC cells and normal human gastric epithelial cells signifi cantly increased and apoptosis decreased, but cell cycle was not significantly altered. After transfection with plasmids expressing TFF, results revealed the proliferation rate of human GC cells and normal human gastric epithelial cells signifi cantly decreased, apoptosis increased, and cells in G and G/M phase decreased, while cells in S-phase increased and cells were arrested in S phase, which suggest that TFF can exert important eff ects on cell proliferation and apoptosis. It is speculated that the inhibition of GC by TFF may be attributed to that it can regulate the balance between cell proliferation and apoptosis. Cell proliferation is promoted and apoptosis rate reduced after silencing TFF expression which breaks the balance of TFF expression. Th e biological characteristics of a tumor are infi nite proliferation and de-diff erentiation. Th e reduced TFF expression may promote the GC development. This speculation is needed to be further confirmed. As a whole, despite a growing number of studies have confi rmed TFF plays a crucial role in the occurrence and development of GC, the exact mechanism of anti-tumor eff ect of TFF is still poorly understood and more studies are required.

CONCLUSION
In our study, we fi nd that TFF can inhibit proliferation and induce apoptosis of GC cells in vitro.

DECLARATION OF INTEREST
Th e authors report no confl icts of interest.