Gastric cancer is one of the leading causes of cancer-associated mortality and has a high tendency to metastasize, making it a priority to develop novel diagnostic and treatment methods at the early stages. The present study investigated the role of a newly-discovered long non-coding RNA, Z38, in gastric cancer cell proliferation, metastasis and apoptosis. It was observed that Z38 was upregulated in tissues from patients with gastric cancer as well as in cultured gastric cancer cells. Knockdown of Z38 decreased the cell proliferative rate, as evidenced by colony formation assays and cell proliferation assays. In addition, Transwell assays and wound-healing assays demonstrated that depletion of Z38 significantly inhibited cell migration and invasion in AGS and MKN74 cells. Furthermore, a cell apoptosis assay and measurement of relative activities of related caspases revealed that depletion of Z38 increased cell apoptosis by promoting the activities of caspase-3 and caspase-9, but not that of caspase-8. Finally, western blot analysis further demonstrated the role of Z38 in the apoptosis of AGS and MKN74 cells. These results suggested that Z38 promotes cell proliferation and metastasis, and inhibits cell apoptosis in gastric cancer. Z38 may represent a novel therapeutic target for the treatment of gastric cancer in clinic.
Gastric cancer is among the most common types of cancer worldwide with an increasing incidence rate each year. Approximately 650,000 patients succumb to gastric cancer, making its mortality rate subsequent to that of lung cancer (
It is known that only 2% of the mammalian genome is able to be translated into protein; however, >85% of the genome exhibits the potential to be transcribed into RNA, while the majority of RNAs serve roles in regulation (
Z38 was a newly-discovered lncRNA by Deng
In the present study, the relative transcript levels of Z38 were examined in patients with gastric cancer and in cultured cells. The roles of Z38 in cell proliferation and metastasis were examined with cell viability assays, colony formation assays and Transwell assays, as well as wound-healing assays. A preliminary study focusing on the effects of Z38 on cell apoptosis was also included. The results of the present study indicated that Z38 may act as a potential therapeutic target for the treatment of gastric cancer.
Gastric cancer tissues and matched adjacent non-cancerous tissues from 100 patients (age range: 35–75 years, average age: 62 years, males: 37, females: 63) who were admitted to the Department of General Surgery, Weifang People's Hospital (Weifang, China) between April 2014 and May 2016, were collected following surgical resection and were immediately frozen in liquid nitrogen. Clinical characteristics of these patients, including age, sex, presenting symptoms and TNM stage were also assessed (
The human gastric cancer KATO III, SGC-7901 and AGS cell lines, as well as the 293T cell line as a control, were purchased from the cell bank of the Chinese Academy of Sciences (Shanghai, China). The human gastric cancer MKN45 and MKN74 cell lines were purchased from American Type Culture Collection (Manassas, VA, USA). All cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.). Primary antibodies against caspase-3 and caspase-9 were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). The primary antibody against GAPDH and the horseradish peroxidase-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).
Total RNA was extracted from human tissues and cultured gastric cancer cells using TRIzol® reagent (Takara Biotechnology Co., Ltd., Dalian, China), according to the manufacturer's protocols. The RNA quality and concentration were determined by collecting the absorbance with the Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Inc.). Reverse transcription (RT) of first-strand cDNAs (1 µg) was performed using PrimeScript RT Master mix (Takara Biotechnology Co., Ltd.), according to the manufacturer's protocol. All PCR amplifications were performed in an ABI PRISM 7900 Real-Time system (Applied Biosystems; Thermo Fisher Scientific, Inc.) with the SYBR® Premix Ex Taq™ kit (Takara Biotechnology Co., Ltd.). The thermocycling conditions were as follows: Initial denaturation at 95°C for 5 min, followed by 45 repeats of a three-step cycling program consisting of 10 sec at 95°C (denaturation), 10 sec at 60°C (primer annealing) and 10 sec at 72°C (elongation), and a final extension step for 10 min at 72°C. The primer sequences used for qPCR are listed in
For knockdown of Z38, specific siRNAs (siZ38-1 and siZ38-2, 1uM) were designed and synthesized by Santa Cruz Biotechnology, Inc. and diluted to a final concentration of 20 mM. The transfection assay was performed using Lipofectamine® 2000 transfection reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocols. At 6 h after transfection, the medium was replaced with fresh Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum. The cells were subject to subsequent analysis 72 h later.
AGS and MKN74 cells were seeded onto 12-well plates 24 h prior to transfection, following which specific siRNA against Z38 was transfected. Subsequently, a total of 500 cells were seeded onto a 6-well plate in each treatment group. The plates were incubated at 37°C for 2 weeks without changing the culture medium and mixed with methanol at room temperature for 15 min. Finally, the colonies were stained with 1% crystal violet for 10 min at room temperature and images were captured (Leica DM IRB; Leica Microsystems GmbH, Wetzlar, Germany) in 5 random fields of view. The whole plates were counted under a Nikon light microscope (×200 magnification) and were statically analyzed.
AGS and MKN74 cells were seeded onto 96-well plates (3,000 cells/well) and were cultured overnight. Cells were subsequently transfected with siZ38-1 or siZ38-2 (10 µM), followed by incubation in DMEM for another 72 h. Cell viabilities were determined for 5 consecutive days using the MTT assay. For this, 2 mg/ml MTT solution was added to each well, followed by incubation for 4 h at 37°C. Subsequently, the medium was removed and 200 µl dimethyl sulfoxide was added to dissolve the purple formazan. The plate was agitated for 5 min at room temperature and the optical density was subsequently determined at 570 nm using a spectrophotometer.
AGS and MKN74 cells were cultured in 24-well plates and transfected with specific Z38 siRNA or control siRNA, with transfection protocols as stated previously. At 48 h post-transfection, cells were harvested and single-cell suspensions in serum-free DMEM were prepared, of which 150 µl (3×104 cells) was seeded into the upper chamber of an 8-mm Transwell plate (Corning Life Sciences, Corning, NY, USA). The lower chamber was filled with 600 µl DMEM, supplemented with 10% FBS. For the invasion assay, the membrane was coated with Matrigel (Beyotime Institute of Biotechnology) 6 h prior to seeding. Following incubation at 37°C for 12 h, cells were fixed with ice-cold methanol for 20 min and stained with 0.1% crystal violet for 5 min at room temperature. Images were captured under an inverted light microscope at a magnification of ×200.
AGS and MKN74 cells were seeded onto 6-well plates (~5×105 cells/well) and transfected with siZ38 or control siRNA. A sterile 10 µl pipette tip was used to scrape across the center of each well at 48 h post-transfection three times and immediately cultured with serum-free medium (Gibco; Thermo Fisher Scientific, Inc.). Cells were allowed to migrate for 12 h, following which scratches were observed and images. Subsequently, cells were rinsed with phosphate-buffered saline were captured for each group using a light microscope (×200 magnification). Each assay was performed in triplicate and repeated at least three times.
The Annexin V/propidium iodide (PI) assay was performed according to the manufacturer's protocols (Invitrogen; Thermo Fisher Scientific, Inc.). In brief, AGS and MKN74 cells were plated onto 6-well plates and transfected with control or specific siRNA against Z38. Subsequently, cells were washed with pre-cold phosphate-buffered saline, trypsinized and re-suspended in 100 µl binding buffer with 2.5 µl fluorescein isothiocyanate-conjugated Annexin-V and 1 µl PI (100 µg/ml). Cells were subsequently incubated at room temperature for 15 min in the dark. A total of >10,000 cells were collected and calculated using a flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) with FolwJo 7.6.1 software (FlowJo LLC, Ashland, OR, USA).
The activities of caspase-3, caspase-8 and caspase-9 were determined using caspase activity kits (Beyotime Institute of Biotechnology), according to the manufacturer's protocols. In brief, cells were transfected with siRNAs for 72 h. Subsequently, cell lysates were collected by low speed centrifugation (860 × g for 5 min at 4°C). An equal amount of protein (10 µl) from each sample were added to 96-well plates and mixed with an aliquot of 80 µl reaction buffer (Beyotime Institute of Biotechnology) supplied with caspase substrates (2 mM). Following incubation at 37°C for 4 h, caspase activities were determined using a TECAN reader at an absorbance of 450 nm.
Total protein was extracted from cultured cells. Cell lines were permitted to grow until they reached 95% confluence. Following two washes with phosphate-buffered saline, cells were lysed with a general lysis buffer (NP40; Beyotime Institute of Biotechnology) to generate the total protein lysate. The protein was quantified with a Pierce BCA Protein Assay kit (Thermo Fisher Scientific, Inc.). Protein was then subjected to 10% SDS-PAGE by loading equal amounts of whole protein (50 µg) per lane. PVDF membranes were blocked with 5% milk in TBST at room temperature for 1 h and then incubated with the following antibodies at 4°C for overnight: Anti-caspase-3 (cat. no. 9662; dilution, 1:1,000), anti-caspase-9 (cat. no. 9508; dilution, 1:1,000), anti-GAPDH (cat. no. sc-47724; dilution, 1:2000), prior to being incubated with a horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotech., Santa Cruz, USA, dilution, 1:5,000 at room temperature for 1 h). GAPDH was synchronously detected as a loading control. Immunoreactivity was determined using enhanced chemiluminescence autoradiography (Thermo Fisher Scientific., Inc.). Image J 2× software (National Institutes of Health, Bethesda, MD, USA) was used to quantify the western blotting data. Each experiment was repeated at least three times.
Data are expressed as the mean ± standard deviation. A two-tailed Student's t-test was used to compare the means of two groups, while one-way analysis of variance was used for comparisons among multiple groups (≥3 groups), followed by a least significant difference post hoc test. P<0.05 was considered to indicate a statistically significant difference. All experiments were repeated at least three times unless otherwise stated.
In order to investigate the role of Z38 in gastric cancer, the relative transcript level of Z38 in 100 clinical gastric cancer tissues was examined. As demonstrated in
Next, two specific siRNAs against Z38, namely siZ38-1 and siZ38-2, were designed. Subsequently, these two siRNAs were transfected into AGS and MKN74 cells. It was revealed that the relative transcript level of Z38 was significantly decreased by siZ38-1, but not siZ38-2 (
In order to further investigate the role of Z38, Transwell and wound-healing assays were performed. It was revealed that ~250 AGS and 220 MKN74 cells migrated through the membrane in control and siNC-treated groups, while only 120 AGS and 125 MKN74 cells were observed on the lower surface of the membrane in the migration assay (
Increased cell proliferation rate, cell metastasis potential and inhibited cell apoptotic capacity were the main manifestations of the majority of malignancies (
Gastric cancer is the fourth most common type of cancer among males and the sixth among females (
Aberrant expression of certain regulatory RNAs markedly influences cancer origination and progression (
The induction of apoptosis may be divided into two categories: The intrinsic and extrinsic pathways. The initiation of the intrinsic pathway is associated with the pro-apoptotic factors, B cell lymphoma-associated X protein (Bax) and B cell lymphoma-associated death promoter (Bad), which leads to increased permeability of the mitochondrial membrane, loss of membrane potential and release of cytochrome
In conclusion, the present study revealed that the expression of Z38 was upregulated in human gastric cancer. Knockdown of Z38 in AGS and MKN74 cells inhibited cell proliferation and metastasis, and promoted cell apoptosis by upregulating the activities of caspase-3 and caspase-9. The results of the present study indicated the oncogenic potential of Z38 in human gastric cancer and provided evidence that Z38 may serve as a potential therapeutic target for the treatment of gastric cancer.
Not applicable.
No funding received.
The datasets used during the current study are available from the corresponding author on reasonable request.
YW and CZ performed the experiments. XW and TL analyzed the data. RZ, YW and JZ analysed the data and revised the manuscript and references. QH and ZS designed the project, analyzed the data and reviewed the manuscript.
Written informed consent was obtained from each patient and the present study was approved by the Ethics Committee of Weifang People's Hospital (Weifang, China).
The study participants provided written informed consent for the publication of the data included in the present study.
The authors declare that they have no competing interests.
Long non-coding RNA Z38 was overexpressed in human gastric cancer tissues and cells. (A) A total of 100 patients with gastric cancer were included and the expression of Z38 in the tumor tissues and their adjacent non-cancerous tissues was investigated. ***P<0.0001, Cancer vs. Non-cancer. (B) Five gastric cancer cell lines KATO III, MKN45, SGC-7901, AGS and MKN74 and a control cell line 293T were involved to investigate the expression of Z38 using reverse transcription-quantitative polymerase chain reaction analysis. *P<0.05 vs. 293T cells.
Knockdown of Z38 in AGS and MKN74 cells inhibited cell proliferation. (A) Two specific siRNAs against Z38 were transfected into AGS and MKN74 cells and the relative transcript levels of Z38 were examined. (B) Colony formation assay was performed on siZ38 (short for siZ38-2) transfection in AGS and MKN74 cells. *P<0.05, vs. Control in AGS cells; #P<0.05, vs. Control in MKN74 cells. (C) The cell proliferation rate was investigated when AGS cells were transfected with siZ38 or siNC. (D) The cell proliferation rate was investigated when MKN74 cells were transfected with siZ38 or control siNC. *P<0.05, vs. Control. siRNA, small interfering RNA; siNC, negative control siRNA.
Knockdown of Z38 in AGS and MKN74 cells inhibited cell metastasis. (A) Cell migration assays were performed when AGS and MKN74 cells were transfected with siZ38 for 72 h. (B) Cell invasion assays were performed when AGS and MKN74 cells were treated with siZ38 for 72 h. *P<0.05 vs. Control in AGS cells; #P<0.05 vs. Control in MKN74 cells. (C) Representative images of wound-healing assays. (D) Following random selection of 5 fields for each group of cells, the wound closure rate was quantified in siZ38-transfected AGS and MKN74 cells. *P<0.05 vs. Control in AGS cells; #P<0.05 vs. Control in MKN74 cells. siZ38, Z38 small interfering RNA; siNC, negative control small interfering RNA.
Knockdown of Z38 in AGS and MKN74 cells increased the rate of cell apoptosis. (A) Cell apoptotic rates were examined upon siZ38 transfection in AGS and MKN74 cells. (B) The relative activities of caspase-3 were examined upon siZ38 transfection in AGS and MKN74 cells. (C) The relative activities of caspase-8 were examined upon siZ38 transfection in AGS and MKN74 cells. (D) The relative activities of caspase-9 were examined upon siZ38 transfection in AGS and MKN74 cells. *P<0.05 vs. Control in AGS cells; #P<0.05 vs. Control in MKN74 cells. siZ38, Z38 small interfering RNA; siRC, negative control small interfering RNA.
Knockdown of Z38 in AGS and MKN74 cells increased the protein expression levels of caspase-3 and caspase-9. AGS and MNK74 cells were treated with siZ38 for 72 h, and western blot analysis was performed to reveal the protein expression levels of caspase-3 and caspase-9. GAPDH was included as an internal control. Two bands of cl-caspase-3 were detected, but the top bands were specific to cl-caspase-3, the molecular weight of which is 17KD, and the lower one was also from cl-caspase-3, which has a smaller molecular weight (12KD). siZ38, Z38 small interfering RNA; cl-caspase, cleaved-caspase.
Primers sequences used in reverse transcription-quantitative polymerase chain reaction.
Gene | Primer nucleotide sequences |
---|---|
Z38 | |
Forward | 5′-AGTGGGATTGTGGAGACGGTGT-3′ |
Reverse | 5′-AGGTAAAAGGAACTGGCAACGC-3′ |
GAPDH | |
Forward | 5′-GTGGACATCCGCAAAGAC-3′ |
Reverse | 5′-AAAGGGTGTAACGCAACTA-3′ |
Association between Z38 and clinical variables among 100 gastric cancer patients.
Expression of Z38 | ||||
---|---|---|---|---|
Variable | No. | Low (n=40) | High (n=60) | P-value |
Age, years | 0.526 | |||
<40 | 18 | 7 | 11 | |
40–50 | 28 | 16 | 12 | |
>50 | 54 | 27 | 27 | |
Sex | 0.094 | |||
Male | 62 | 29 | 33 | |
Female | 38 | 11 | 27 | |
Presenting symptoms | ||||
Painless lump | 46 | 21 | 25 | 0.159 |
Painful lump | 48 | 15 | 33 | |
Atypical symptoms | 6 | 4 | 2 | |
T, cm | <0.001 |
|||
T1 (≤2) | 34 | 21 | 13 | |
T2 (>2 and <5) | 26 | 12 | 14 | |
T3 (≥5) | 22 | 6 | 16 | |
T4 (any size with distant metastasis) | 18 | 1 | 17 | |
N | <0.001 |
|||
N0 | 44 | 28 | 16 | |
N1 or above | 56 | 12 | 44 | |
Distant metastasis (M) | 0.023 |
|||
M0 | 45 | 24 | 21 | |
M1 | 55 | 16 | 39 | |
TNM stage | 0.008 |
|||
I/II | 42 | 19 | 23 | |
III/IV | 58 | 11 | 47 |
P<0.05. T, tumor size; N, lymph node metastasis; M, distant metastasis; TNM, Tumor-Node-Metastasis.