Significance of gastrin‑releasing peptide in ovarian cancer ES2 cells

  • Authors:
    • Yanyan Jia
    • Huirong Shi
    • Dongmei Fan
  • View Affiliations

  • Published online on: May 20, 2015     https://doi.org/10.3892/ol.2015.3240
  • Pages: 359-363
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Abstract

The present study aimed to investigate the effect of gastrin‑releasing peptide (GRP) on the proliferation and invasion of ovarian cancer ES2 cells. The ovarian cancer ES2 cells were transfected with small interfering RNA against GRP. Cell proliferation was assessed using the Trypan blue assay, apoptosis was determined using propidium iodide/fluorescein isothiocyanate and flow cytometry, and the invasion ability was detected using the Transwell assay. The results revealed that the expression of GRP significantly decreased following transfection with GRP‑short hairpin RNA. Furthermore, the silencing of GRP resulted in increased apoptosis and a reduced invasive ability of the ES2 cells. It was concluded that GRP may regulate the proliferation and migration of human ovarian cancer cells, which indicates that GRP may be a potential novel target for the treatment of ovarian cancer.

Introduction

Gastrin-releasing peptide (GRP) is a 27-amino acid peptide, which was first identified as a cytokine secreted from P cells in the alimentary tract. The local secretion of GRP regulates gastrointestinal hormones and gallbladder contraction (1). Previous studies revealed that GRP and the GRP receptor (GRPR) were overexpressed not only in the gastrointestinal tract, but also in a number of other malignant tumors and cell lines (25). It has been established that epithelial cells from regions other than the parenteral tract can also excessively secret GRP under the regulation of carcinogenic factors. GRP can then bind to the GRPR and promote the mitosis of cells.

We previously screened samples of serum, ascites and cyst fluid from 30 ovarian carcinoma patients by matrix-assisted laser desorption/ionization time-of-flight mass spectrometer technology combined with WCX magnetic beads. The results indicated that the elevated peptide peaks at 2881 and 2897 Da may correspond to the same GRP polypeptide, and confirmed that GRP may be involved in the carcinogenesis of ovarian cancer (6). The biological effects of GRP in colorectal, gastric and prostate cancer have been previously reported (79). However, studies concerning the biological effects of GRP in ovarian cancer have yet to be conducted.

In the present study, a small interfering RNA (siRNA) against GRP was constructed (10) and transfected into human ovarian cancer ES2 cells, which are known to highly express GRP. The biological changes in the ES2 cells were then investigated.

Materials and methods

Materials

The Escherichia coli (E. coli; DH5α) and pGenesil-1.1dBm2 vector were purchased from the Wuhan JinSai Biological Technology Engineering Company (Wuhan, China). Monoclonal mouse anti-human GRP (dilution, 1:1,000) and mouse anti-human β-actin (dilution: 1:1,000) antibodies were obtained from R&D Systems, Inc., (Minneapolis, MN, USA). The human ovarian cancer ES2 cell line was supplied by the China Center for Type Culture Collection at Wuhan University (Wuhan, China).

Cell culture

The ES2 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 µg/ml streptomycin and 100 U/ml penicillin. The cell cultures were maintained in a humidified atmosphere of 5% CO2 at 37°C in an incubator.

Design of short hairpin (sh)RNA construction for GRP

According to the GRP complementary (c)DNA sequence obtained from Genbank, the GRP-targeted shRNA construct was designed using the RNAi software from Ambion Life Technologies (Carlsbad, CA, USA). The cDNA target sequence was 192–210 bp in length. The oligonucleotide was synthesized by Wuhan JinSai Biological Technology Engineering Company, and the sequence of the GRP-shRNA was as follows (the guide peptide is in bold): Sense, 5′-CACCACCGTGCTGACCAAGATGTTTCA AGACGACATCTTGGTCAGCACGGTTTTTTTG-3′; and antisense, 5′-AGCTCAAAAAAACCGTGCTGACCAAGA TGTCGTCTTGAAACATCTTGGTCAGCACGGT-3′.

Construction and cloning of pGenesil-GRP-shRNA

The GRP-shRNA was inserted into the SacI site of the expression vector, pGenesil-1.1dBm2. The product of the recombination reactions, pGenesil-GRP-shRNA, was used to transform the competent E. coli strain, DH5α, during the transformation assay. Briefly, the bacteria were washed with a 0–100 mM CaCl2 solution, resuspended in 200 µl of the same solution, and then placed on ice for 1 h. Next, the pGenesil-GRP-shRNA plasmid DNA was added for 30 min. The sample was then incubated at 42°C for 90 sec. Following the addition of 1 ml Luria-Bertani broth, the sample was incubated for 1 h at 37°C. In total, 100 µl of the transformation mixture was transferred onto a Luria-Bertani agar plate containing 100 µg/ml kanamycin and 100 µg/ml neomycin. The transformation frequency was determined by calculating the ratio of the number of transformants per viable cell per milliliter.

The plate was then cultured at 37°C for 16–24 h, the positive colonies were amplified in Luria-Bertani broth and the plasmid was extracted using a plasmid extraction kit (Qiagen GmbH, Hilden, Germany) The plasmid was identified by electrophoresis and restriction enzyme digestion.

pGenesil-GRP-shRNA transfection

An equal number of ES2 cells (0.8×106 per well) were seeded into 35-mm dishes. The cells were left for 24 h to attach and reach 70–80% confluency. The transfection assay was performed using Lipofectamine 2000 transfection reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacturer's instructions. Untransfected cells and cells transfected with an empty vector were used as the controls.

The efficiency was assessed according to the concentration of GFP at 24 h post-transfection. The cells were harvested 2–3 days after transfection, and then cultured in complete medium containing 800 µg/ml G418 for 7–8 days. Fresh medium was added every 3 days until the untransfected control cells were all dead. Next, the cells were cultured in complete medium containing 200 µg/ml G418. The positive clones appeared ~20 days later. These positive cells were recultured in fresh bottles and designated pGenesil-GRP-shRNA cells or pGenesil-1.1dBm2 cells.

Reverse transcription polymerase chain reaction (RT-PCR)

The total RNA was extracted from the positive ES2 cell clones using TRIzol (Invitrogen Life Technologies), and then reversed into cDNA by the Super SCRIPTM cDNA kit from Takara Biotechnology Co., Ltd. (Dalian, China). In order to examine the expression of endogenous GRP mRNA, the RT-PCR was performed using the following primers: GRP forward, 5′-AGAGTACATCAGGTGGGA-3′ and reverse, 5′-CAGAAG ATGCTGCTTTAAAA-3′ (product size, 375 bp); and β-actin forward, 5′-ACTCTTCCAGCCTTCCTTC-3′ and reverse, 5′-AATCCTGAGTCAAGCCAAA-3′ (product size, 490 bp). β-actin was used as the internal reference. The amplification conditions were as follows: 94°C for 5 min, followed by 30 cycles at 94°C for 40 sec, 60°C for 30 sec and 72°C for 30 sec, and finally 72°C for 5 min. The PCR products were electrophoresed in 1.5% agarose gel, stained with ethidium bromide and then analyzed using the Q550IW image analysis system (Leica Imaging Systems, Cambridge, UK). The integral optical density of each band was calculated according to the following equation: Integral absorbance = average absorbance × area.

Western blotting

The positive ES2 cell clones were resuspended in SDS loading buffer, heated for 10 min at 100°C, and then centrifuged at 13,000 × g for 15 min at 4°C. The supernatants were collected and separated by SDS-PAGE electrophoresis, and then transferred to a polyvinylidene difluoride membrane. The membrane was dipped in 5% skimmed milk powder for 2 h in order to block non-specific binding sites, and then incubated with GRP or β-actin primary antibodies at 4°C overnight. Subsequent to rinsing three times in Tris-buffered saline and Tween 20 (TBST), the membrane was incubated with the corresponding secondary antibody labeled with horseradish peroxidase for 2 h, and then rinsed in TBST a further three times. Next, the membrane was incubated with SuperSignal West Dura Chemiluminescent Substrate (Thermo Fisher Scientific) for 5 min. Finally the images were obtained by X-ray film exposure.

Cell proliferation detection

The untransfected cells and the ES2 cells transfected with pGenesil-GRP-shRNA or pGenesil-1.1dBm2 vector were cultured. The percentage of dead cells was determined by staining with Trypan blue each day for 7 days.

Flow cytometric analysis of apoptosis

The ES2 cells were collected and washed once with phosphate-buffered saline and then resuspended in 1×106/ml annexin-binding buffer. In total, 100 µl of the cell solution was stained with 5 µl Annexin V and 5 µl propidium iodide for 15 min. The stained cells were analyzed by FACScan flow cytometry (BD Biosciences, Franklin Lakes, NJ).

Cell invasion

The Transwell assay was performed using a commercial kit purchased from Corning Inc. (Corning, New York, NY, USA), as previously described (11). Briefly, collagen-coated membranes were seeded with 1×105 cells in 500 µl DMEM. In all instances, cell migration was assessed 5 h after plating, as recommended by the manufacturer.

Statistical analysis

The data are expressed as the mean ± standard deviation and were analyzed using SPSS 13.0 (SPSS, Inc., Chicago, IL, USA). Student's t-test was performed for comparison between two groups. P<0.05 was used to indicate a statistically significant difference.

Results

Successful construction and transfection of pGenesil-GRP-shRNA

A SacI restriction site was designed at each end of the GRP-shRNA fragment. The fragment was inserted into the vector, pGenesil-1.1dBm2, which was digested by SacI. As shown in Fig. 1, when the pGenesil-GRP-shRNA plasmid was digested by SacI, a 916-bp band appeared, which corresponded to GRP-shRNA. The pGenesil-GRP-shRNA plasmid was sequenced, and it was confirmed that pGenesil-GRP-shRNA had been successfully constructed.

For the transfection assays, the cells were screened using the G418 antibiotic; only the successfully transfected cells were able to survive. The pGenesil-1.1dBm2 vector carried a green fluorescent protein, which emitted green fluorescence under light excitation. As shown in Fig. 2, the positive clones of ES2 appeared green under the laser.

GRP silencing by GRP-shRNA

As shown in Fig. 3, the expression level of GRP was calculated by the integral optical density of the band. The mRNA level of GRP in the transfected cells was significantly lower than that of the control transfected and untransfected groups (0.36, 0.87 and 0.91, respectively; P<0.05). The protein level of GRP was also markedly decreased in the transfected group compared with the control transfected and untransfected groups (0.13, 0.47 and 0.49, respectively; P<0.05; Fig. 4). The results revealed that the ES2 cells were successfully transfected by pGenesil-GRP-shRNA and that the expression of GRP was significantly silenced.

GRP silencing inhibits proliferation

The ES2 cells were transfected with GRP-shRNA or vector alone, untransfected cells were used as the control. The cells were then cultured for 5 days and the cell viability was detected each day. The results demonstrated that the ES2 cells transfected with GRP-shRNA had a significantly lower proliferation ability compared with the control (P<0.05, Fig. 5), while there was no observed difference in the proliferation ability of the ES2 cells transfected with the vector alone compared with the control (P>0.05) (Fig. 5).

GRP silencing induces apoptosis

As shown in Fig. 6, following transfection with pGenesil-GRP-shRNA, the apoptosis rate of the ES2 cells significantly increased (from 8.69 to 31.47%), while that of the vector group increased only slightly (from 8.69 to 11.47%); a significant difference was observed between the two groups (P<0.05).

Effects of 3.6 GRP siRNA on the invasion ability of ES2 cells

Cells that had migrated over the microporous membrane were stained by methylene blue and were counted under an inverted microscope (IX71; Olympus Corporation, Tokyo, Japan) (Fig. 7). The invasive ability of the cells (the number of cells that had migrated over the microporous membrane) in the transfected group was 21±5.3, which was significantly lower than that of the control groups (49±11.8 for the vector transfected group; 43±10.1 for the untransfected group) (P<0.05). The results suggested that silencing of GRP significantly weakened the invasive ability of ES2 cells, and revealed that the GRP may be important for the invasion of ovarian cancer.

Discussion

GRP and its receptor, GRPR, are overexpressed in a number of different tumors, and are believed to have an important role in carcinogenesis and tumor progression (12). It has been hypothesized that the carcinogenic ability of GRP relies on its affinity to GRPR. It was previously reported that in neurofibromatosis, silencing of GRPR through specific siRNA resulted in a significant reduction in proliferation and cell migration, and was accompanied by the inactivation of Akt and the activation of phosphatase and tensin homolog, which is a known inhibitor of the phosphoinositide 3-kinase/Akt (PI3K/Akt) pathway. This indicated that the PI3K/Akt signaling pathway may be regulated by GRP/GRPR (13). Another study concerning small cell lung cancer indicated that the overexpression of GRP led to an elevated level of Akt, which is known to be the initiating factor of the epidermal growth factor receptor (EGFR) signaling pathway (14). As a high level of EGFR is considered to contribute to the resistance of small lung cancer to gemcitabine, the results of the present study indicate that GRP may also have a role in chemotherapy resistance through activating the EGFR signaling pathway in tumors.

In the present study, the expression of GRP in the ES2 cells was downregulated using siRNA technology. The results revealed that the protein level of GRP significantly decreased following transfection, which was accompanied by a decrease in the proliferation and migration ability of ES2 cells, and a marked increase in the rate of apoptosis. This suggested that GRP has a role in the proliferation and cell migration of human ovarian cancer cells. This finding is consistent with that of previous studies concerning other types of tumor (1518). Ischia et al (15) reported that GRP and GRPR are overexpressed in prostate cancer and can stimulate the growth of prostate cancer cells. In addition, GRP was found to be associated with the migration and metastasis of cancer cells in neuroblastoma and breast cancer (1618). It can therefore be concluded that GRP may be a potential novel target for the treatment of ovarian cancers.

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Spandidos Publications style
Jia Y, Shi H and Fan D: Significance of gastrin‑releasing peptide in ovarian cancer ES2 cells. Oncol Lett 10: 359-363, 2015
APA
Jia, Y., Shi, H., & Fan, D. (2015). Significance of gastrin‑releasing peptide in ovarian cancer ES2 cells. Oncology Letters, 10, 359-363. https://doi.org/10.3892/ol.2015.3240
MLA
Jia, Y., Shi, H., Fan, D."Significance of gastrin‑releasing peptide in ovarian cancer ES2 cells". Oncology Letters 10.1 (2015): 359-363.
Chicago
Jia, Y., Shi, H., Fan, D."Significance of gastrin‑releasing peptide in ovarian cancer ES2 cells". Oncology Letters 10, no. 1 (2015): 359-363. https://doi.org/10.3892/ol.2015.3240