Contributed equally
This study investigated the role of miR-214 in modulating proliferation and invasion of human colon cancer SW620 cells. Fifty-five patients with colon cancer who were treated in China-Japan Union Hospital of Jilin University from March 2014 to March 2015 were enrolled into this study. Their cancer and corresponding paracancerous tissues were collected and the expression levels of miR-214 were determined by RT-qPCR. A miR-214 expression vector was constructed. SW620 cells were transfected with the miR-214 expression vector and a blank vector. Cells transfected with the miR-214 expression vector were assigned to the miR-214 positive group and cells transfected with the blank vector were assigned to the miR-214 negative group. Cell proliferation, invasion and apoptosis were assessed by MTT assay, Transwell migration assay and TUNEL apoptosis assay, respectively. The RT-qPCR results showed that the expression level of miR-214 in colon cancer tissue, as well as in miR-214 negative cells, was significantly lower than that in paracancerous tissue (P<0.05 for both). In cell comparison, the expression level of miR-214 in the miR-214 positive group was significantly higher than that in the miR-214 negative group (0.483±0.001 vs. 0.172±0.001; P<0.05). The proliferation level of SW620 cells in the miR-214 positive group was lower than that in the miR-214 negative group (P<0.05). The Transwell migration assay indicated that there were less cells penetrating the membrane in the miR-214 positive group than in the miR-214 negative group (P<0.05). In addition, The apoptosis rate of cells in the miR-214 negative group was significantly lower than that in the miR-214 positive group (P<0.05). Finally, the low expression of miR-214 was found in colon cancer, indicating that miR-214 is a cancer suppressor playing an opposing role in colon cancer onset and progression. Therefore, miR-214 can promote apoptosis of colon cancer cells SW620 by inhibiting their proliferation and invasion.
Colon cancer is the most common malignant tumor of the digestive tract. It often occurs among adults aged above 40 years with a male-to-female ratio of 2–3:1. Colon cancer is a leading cause of cancer deaths worldwide (
MicroRNAs (miRNAs) are widely expressed in eukaryotic organisms, regulating cell proliferation, differentiation and apoptosis. Aberrations in the process of miRNA biosynthesis were associated with a variety of pathophysiological processes (
In this study, the expression levels of miR-214 in cancer and paracancerous tissues collected from patients with colon cancer were analyzed. The role of miR-214 in modulating proliferation and invasion of human colon cancer SW620 cells was explored as well. The goal of this study was to provide a new theoretical basis for the screening, diagnosis and treatment of colon cancer.
A total of 55 patients with colon cancer were enrolled in this study and they were treated in China-Japan Union Hospital of Jilin University (Changchun, China) from March 2014 to March 2015. Their cancer and corresponding paracancerous tissues were collected. The patients, including 31 males and 24 females, were aged 35–70 years with an average age of 55.43±12.75 years. Patients who were pathologically diagnosed with colon cancer and met the following criteria were included in this study: i) without liver, kidney and other organ dysfunction before surgery; and ii) without bleeding and clotting disorders before surgery. Patients who met the following criteria were excluded from this study: i) being treated prior to this study; ii) with too large tumor; and iii) with other diseases such as lung or chest wall diseases.
This study was approved by the Ethics Committee of China-Japan Union Hospital of Jilin University. The patients or their families signed an informed consent.
Human colon cancer SW620 cells were purchased from Shanghai Beinuo Biotechnology Co., Ltd. (Shanghai, China) and cultured in Leibovitz's L-15 medium purchased from Changzhou Beiyuanxin Biotechnology Co., Ltd. (Jiangsu, China). The cell culture was maintained at constant 37°C and pH 6.8–7.4 in an incubator supplied with 5% CO2. A miR-214 expression vector was constructed by Shanghai GenePharma Co., Ltd. (Shanghai, China). The constructed miR-214 expression vector (miR-214 positive group) and blank vector (miR-214 negative group) were cultured, respectively, with trypsin-digested human colon cancer cells SW620 in Leibovitz's L-15 medium at 37°C in an incubator supplied with 5% CO2 for 24 h. After transfection, the cells in the two groups were subjected to analytical experiments.
Total RNA in cancer and paracancerous tissues was extracted by using TRIzol reagent purchased from Shanghai Mingjing Biotech Co., Ltd. (Shanghai, China), following the instructions contained in the TRIzol kit. The concentration and purity of the extracted RNA were measured with an MD1000 Microscale UV Spectrophotometer manufactured by Thmorgan Biotechnology Co., Ltd. (Beijing, China). Integrity of the RNA was assessed by 3% agarose gel electrophoresis. The gel electrophoresis set was purchased from Shanghai Jingke Science and Technology Co., Ltd. (Shanghai, China).
Synthesis of cDNA from the extracted total miRNA was performed, via reverse transcription, at 37°C for 45 min and 95°C for 5 min by using the fluorescence quantitative PCR kit purchased from Thermo Fisher Scientific Co., Ltd. (Shanghai, China) following the instructions contained in the kit. Total volume of the PCR amplification reaction system was 20 µl. The PCR reaction was performed as follows: pre-denaturation at 95°C for 10 min, 40 cycles of 95°C for 10 sec (denaturation), 60°C for 20 sec (annealing), and 72°C for 10 sec (extension), followed by extension at 72°C for 5 min at the end of the cycles. U6 was used as the reference gene. All samples were run in triplicate. The results were analyzed by using the 2−ΔΔCq method (
The transfected human colon cancer SW620 cells in the miR-214 positive and negative groups were prepared into a single cell suspension, followed by routine seeding in a 96-well cell culture plate. After culturing for 6 h, 20 µl of MTT solution (5 mg/ml) was added to a portion of the cultured cells, and incubation was continued at 37°C for 4 h. After sucking away the supernatant containing the medium and reagents, DMSO was added, followed by shaking on a rocking platform shaker for 15 min. The absorbance at a wavelength of 570 nm was measured by using an ELISA reader. After culturing for 12, 24, 48 and 72 h, the above MTT assay was repeated to obtain data at different time-points. The MTT assay kits were purchased from Shanghai LMAI Bio Co., Ltd (Shanghai, China).
Single cell suspensions of transfected SW620 cells in the miR-214 positive and negative groups were prepared and seeded in triplicate (~1×105−1×106 cells per well) into Transwell upper chambers, respectively. The number of cells passing through the membrane in each well was counted two weeks later. The Transwell plates were purchased from Shanghai Yuanzi Biotechnology Co., Ltd. (Shanghai, China).
After culturing for 48 h, the cells (approximately 5×107 cells/ml) were fixed in 4% neutral formalin for 10 min at room temperature. Following removal of the supernatant, the fixed cells were washed twice with PBS for 5 min each. The cells were then incubated in PBS containing 2% hydrogen peroxide for 5 min at room temperature. Following removal of the supernatant, the fixed cells were washed with PBS twice for 5 min each, and were then stained by using TUNEL kits (Shanghai Runwell Technology Co., Ltd., Shanghai, China) according to the protocol contained in the kit. The number of TUNEL-positive cells in five 400-fold fields of view was counted by using the image analysis software Image-pro Plus 5.0. The cumulative optical density value represented the total number of TUNEL positive cells. The measurement was repeated three times.
The statistics software SPSS 19.0 (AsiaAnalytics Formerly SPSS China, Shanghai, China) was used for statistical analysis. The χ2 test was applied to rate comparison. Measurement data were expressed as mean ± standard deviation. The t-test was used for comparison of data that were normally distributed. P<0.05 was considered to indicate a statistically significant difference.
The levels of miR-214 in colon cancer and paracancerous tissues, as well as in SW620 cells, were measured by RT-qPCR. As shown in
The expression level of miR-214 was not associated with the patient's sex, age, clinical stage, depth of tumor invasion, degree of differentiation, or lymph node metastasis (P>0.05). However, the miR-214 level was associated with histological classification of colon cancer. As shown in
The proliferation of SW620 cells in the miR-214 positive and negative groups was determined by MTT assay. As shown in
The invasiveness of SW620 cells in the miR-214 positive and negative groups was measured by Transwell
Apoptosis of SW620 cells in the miR-214 positive and negative groups was measured by TUNEL apoptosis assay. As shown in
Research focusing on miRNAs represents one of the most popular topics in cancer research. The aberrant expression of miRNAs can modify the expression of tumor-related genes, impacting tumor onset and progression (
In order to further study the expression of miR-214 in colon cancer and its biological effect on colon cancer, human colon cancer SW620 cells were transfected with a miR-214 expression vector. The transfected cells were cultured and used in the study of miR-214 biological effects by MTT proliferation, Transwell
Wang
In conclusion, miR-214 was downregulated in colon cancer. It exhibited inhibitory effect in the onset of colon cancer as a tumor suppressor gene. The proliferation and invasion of colon cancer SW620 cells were attenuated by miR-214, while the apoptosis rate was increased. It is possible that miR-214 can be a new target in the treatment of colon cancer.
Not applicable.
No funding was received.
The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.
HN wrote the manuscript and extracted total RNA from tissues. DN performed the RT-qPCR. LM contributed to performing the MTT assay. All authors have read and approved the final manuscript.
The study was approved by the Ethics Committee of China-Japan Union Hospital of Jilin University (Changchun, China). Patients who participated in this research, signed an informed consent and had complete clinical data.
Not applicable.
The authors declare that they have no competing interests.
Levels of miR-214 in colon cancer and paracancerous tissues and in human colon cancer SW620 cells measured by RT-qPCR. The RT-qPCR assay showed that the expression levels of miR-214 in colon cancer tissue (0.203±0.001) and in SW620 cells in the miR-214 negative group (0.172±0.001) were comparable (P>0.05), and both were significantly lower than that in the paracancerous tissue (0.515±0.002). The differences were statistically significant (P<0.05). The miR-214 expression level in SW620 cells in the miR-214 positive group (0.483±0.001) was significantly higher than those in SW620 cells in the miR-214 negative group and in the cancer tissue, but was lower than that in paracancerous tissue (P<0.05).
Proliferation of human colon cancer SW620 cells assessed by MTT assay. The MTT assay showed that the OD values of cells in the miR-214 positive group were lower than those in the miR-214 negative group at five time-points of 6, 12, 24, 48 and 72 h, indicating that the proliferation rate of cells in the miR-214 positive group was lower due to the higher miR-214 level. The difference was statistically significant (P<0.05). OD, optical density.
Invasion of human colon cancer SW620 cells measured by Transwell
Apoptosis of human colon cancer SW620 cells measured by TUNEL apoptosis assay. TUNEL apoptosis assay showed that the apoptosis rate of cells in the miR-214 negative group was significantly lower than that in the miR-214 positive group. The difference was statistically significant (P<0.05).
Association of miR-214 expression level with clinicopathological characteristics by χ2 test.
Clinicopathological characteristics | n (%) | miR-214 level | χ2 | P-value |
---|---|---|---|---|
Patient number | 55 | 0.203±0.001 | ||
Sex | 1.087 | 0.282 | ||
Male | 31 (56.36) | 0.212±0.003 | ||
Female | 24 (43.74) | 0.206±0.007 | ||
Age (years) | 1.174 | 0.246 | ||
<45 | 19 (34.55) | 0.203±0.002 | ||
≥45 | 36 (65.45) | 0.211±0.005 | ||
Clinical stage | 0.924 | 0.359 | ||
T1/T2 | 20 (33.36) | 0.203±0.009 | ||
T3/T4 | 35 (62.64) | 0.207±0.001 | ||
Invasion depth | 1.962 | 0.055 | ||
Muscularis | 14 (25.45) | 0.214±0.005 | ||
Serosa | 41 (74.55) | 0.232±0.001 | ||
Differentiation | 1.394 | 0.169 | ||
Well | 43 (78.18) | 0.229±0.006 | ||
Moderately and poorly | 12 (21.82) | 0.214±0.007 | ||
Lymph node status | 1.998 | 0.051 | ||
Metastasis | 32 (58.18) | 0.221±0.006 | ||
No metastasis | 23 (41.82) | 0.202±0.003 | ||
Histological classification | 34.42 | <0.001 | ||
Mucinous carcinoma | 15 (27.27) | 0.116±0.007 | ||
Non-mucinous carcinoma | 40 (72.73) | 0.303±0.004 |