MiR-1-3p inhibits the proliferation and invasion of bladder cancer cells by suppressing CCL2 expression

Abstract

We attempted to analyze the effects of miR-1-3p and CCL2 on the proliferation, migration, and invasion of bladder cancer cells. A total of 18 pairs of bladder cancer tissues with corresponding adjacent tissues and the 6 cases of normal tissues were collected. The expressions of miR-1-3p and CCL2 in the cancer tissues were evaluated using quantitative real-time polymerase chain reaction and western blot. The relationship between miR-1-3p and CCL2 was assessed using luciferase reporter assay. The UM-UC-3 bladder cancer cells were transfected with CCL2 small interfering RNA and miR-1-3p mimics. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, colony formation assay, wound healing assay, Transwell assay, and the flow cytometry test were used to detect the proliferation, migration, invasion, and apoptosis of bladder cancer cells. Bladder cancer tissues had lower levels of miR-1-3p but higher levels of CCL2 than normal tissues (p < 0.05). The transfection of miR-1-3p mimics and CCL2 small interfering RNA remarkably suppressed cell proliferation and invasion and promoted apoptosis of cells (p < 0.05). Results of the luciferase reporter gene assay demonstrated that miR-1-3p targeted CCL2. MiR-1-3p suppresses the proliferation and invasion of urinary bladder cancer cells by targeting CCL2.

Keywords

MiR-1-3p CCL2 urinary bladder cancer

Introduction

Bladder cancer (BC) is the uncontrollable multiplication of urinary bladder cells.¹ As suggested by the National Cancer Institute, BC ranks in the top 10 most prevalent cancers worldwide.^2–4 The annual incidence of new BC cases has been declining over the last 10 years, and the fatality rate has remained stable in most Western countries.⁵ The causes of BC are diverse and complicated. Smoking, however, is the confirmed top cause.^5–7 The overall 5-year survival rate is approximately 75%, and a significant influence factor is the time of diagnosis.⁸ A popular current research topic is targeted therapy. Targeted therapy acts on the genetic pathways responsible for BC,⁴ which is regarded as the next-generation therapy for the disease with the help of genomic and proteomic techniques.^3,9

MicroRNA (miR) is a short non-coding RNA sequence which plays an important role in adjusting gene expressions. It binds to the 3′ untranslated region (UTR) of target messenger RNAs (mRNAs) and affects the corresponding transcription process.^4,10 The aberrant expression of miRs is often observed in various human malignancies and growing processes, suggesting that miRs play a pivotal role in the metastasis and invasion of carcinoma (mostly working as oncogene suppressors).^8,11,12 For instance, Wu et al.¹³ indicated that miR-429 can attenuate the migratory and invasive ability of BC cells. Another study conducted by Xie et al.¹⁰ demonstrated that miR-1 is associated with the viability, migration, and invasion of gastric cancer cells.

Chemokines can mainly be categorized into two groups according to the position of cysteine residues in the primary amino acid sequence –CXC (or α-chemokine) and CC (or β-chemokine).^14–17 Chemokines are critical to the inflammatory response of humans and are secreted to recruit cells from the immune system in response to viral infection and allergic disorders.¹⁸ Some chemokines are even suggested to be involved in modulating the migration of normal cells. Scientists have recently discovered that the deactivation of chemokines may implicate the initiation or progression of a variety of tumors.^19–21 For example, Miyake et al.¹⁸ discovered that the higher expression of CXCL1 is associated with the aggressiveness of human BC. Researches have been carried out to reveal the effects of CXCs and CCs on malignant tumors. As a member of the chemokine family, CCL2 has a connection with BCs; however, little emphasis on this connection has been studied thus far. A study carried out by Chiu et al.²² in 2012 suggested that the elevated expression of CCL2 in BC patients can mediate tumor invasion. Considering that this trend is associated with advanced tumorigenesis, it is worthwhile to confirm their results through further studies.

Our microarray assay illustrated that CCL2 is overexpressed in BC cells, the level of miR-1-3p in cancer tissues is lower than that in normal tissues, and there is a miR-1-3p binding site at CCL2 3′ UTR. These results lead us to hypothesize that CCL2 is a target of miR-1-3p. Considering that relevant papers are scarce, this study aims to further verify that miR-1-3p is able to inhibit the proliferation, metastasis, and invasion of bladder malignancy by suppressing the synthesis of CCL2. We also aim to confirm the target relationship between CCL2 and miR-1-3p.

Materials and methods

Clinical specimens and cell culture

Tissues were collected from BC patients who were undergoing surgery at The Sixth People’s Hospital of Yancheng City during 2014 and 2016. A total of 18 pairs of BC tissues and the corresponding adjacent tissues were collected. Another six cases of normal bladder tissues were also obtained from patients undergoing cystolithotomy or transurethral resection of prostate (TURP). These patients consisted of 17 males and 7 females who had an average age of 56 years. According to the tumor, node, metastasis (TNM) 2010 staging system of BC,²³ these 18 tissues were divided into six T1 phase cases, five T2a phase cases, and seven T2b phase cases. The pathological classification criteria used were in accordance with the standard of urothelial carcinoma formulated by the World Health Organization (WHO)²⁴ and the International Society of Urological Pathology (ISUP). BC adjacent tissues were obtained from areas about 2 cm away from tumor lesions. The use of tissue samples was approved by the ethics committee of The Sixth People’s Hospital of Yancheng City.

The human BC cell lines 5637, T24, and UM-UC-3 were purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences. The primary culture of normal cells was collected from normal bladder tissues. All cells were seeded into 24-well plates with Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco Company, Grand Island, NY, USA) and 10% fetal bovine serum (FBS; Gibco Company) and cultured at 37°C with 5% CO₂.

RNA isolation and real-time polymerase chain reaction

Total RNAs were extracted from tissue samples and 1 mL of TRIzol was added. After being reverse transcribed into complementary DNA (cDNA) using the polymerase chain reaction (PCR) application, extracted RNAs were detected using real-time polymerase chain reaction (RT-PCR). The relative expressions were presented by 2^–DDCt, with U6 as the internal control for miRNA (miR) detection and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as the internal control for CCL2 mRNA detection. Primer sequences are shown in Table 1.

Table 1.

Primer sequences of real-time PCR.

cDNA	Forward primer	Reverse primer
miR-1-3p	5′-TGGAATGTAAAGAAGTATGTAT-3′
CCL2	5′-CAATCAATGCCCCAGTCACC-3′	5′-CCTGAACCCACTTCTGCTTG-3′
U6	5′-CTCGCTTCGGCAGCACA-3′	5′-AACGCTTCACGAATTTGCGT-3′
GAPDH	5′-TGGTATCGTGGAAGGACTCAT-3′	5′-GTGGGTGTCGCTGTTGAAGTC-3′

cDNA: complementary DNA; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Western blot analysis

Total protein was isolated from samples, and protein concentration was detected using bicinchoninic acid (BCA) protein assay.²⁵ After protein electrophoresis, the membranes were blocked with 5% skimmed milk at room temperature for 4 h. The membrane was then incubated for 1 h at 37°C with primary antibodies against CCL2 (1:400; Sigma-Aldrich, St. Louis, MO, USA) and GAPDH (1:800; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and washed four times with Tris-buffered saline with Tween (TBST). Subsequently, the membranes were incubated at 37°C for 1 h with goat anti-rabbit IgG secondary antibodies (1:200; Santa Cruz Biotechnology) with horseradish peroxidase (HRP) and rewashed four times with TBST. The film was developed after the enhanced chemiluminescence (ECL) developing liquid (Millipore, Billerica, MA, USA) reacted with the membranes and the film for 5 min. Images were captured after exposure in a darkroom.

Dual luciferase reporter assay

The CCL2 3′UTR sequence was amplified using RT-PCR and inserted into the pmirGLO luciferase vector (Promega Corporation, Madison, WI, USA) named as CCL2 wt. The complementary sequences for miR-1-3p at CCL2 3′UTR in the mutant-type plasmid, named as CCL2 mut, were mutated using the site mutation method. A luciferase carrier was directly transfected into the UM-UC-3 cells along with miR mimics or negative control plus Lipofectamine™ 2000 (Invitrogen Corporation, Carlsbad, CA, USA). The dual luciferase reporter assay system (Promega Corporation) was used to measure the luciferase activity in cells 48 h after the transfection.

Transfection

UM-UC-3 cells were transfected with miR-1-3p mimics and CCL2 small interfering RNA (siRNA) using the Lipofectamine 2000 reagent. Cells were divided into four groups: a control group (cells without any transfection), an NC group (cells transfected with a nonsense sequence), a mimics group (cells transfected with miR-1-3p mimics), and a siCCL2 group (cells transfected with CCL2 siRNA).

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay

The transfected cells were digested using pancreatic enzymes and seeded into 96-well plates (5 × 10³ cells/well). After incubation for 24, 48, and 72 h, respectively, the cells were stained with 0.5 mg/mL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) for 4 h. The supernatant layer was discarded, and the precipitate was dissolved by adding 200 µL dimethylsulfoxide. The optical absorbance of samples was measured at 490 nm with an enzyme-linked immunosorbent assay (ELISA) reader.

Colony formation assay

The transfected cells were digested using pancreatic enzymes and then suspended into a single-cell suspension where the number of cells was counted and the concentration was adjusted. Subsequently, the suspension was placed into a six-well plate. We ensured that each well had 150 cells and that each well was filled with 3 mL of complete media. After 2 weeks of cultivation at 37°C in 5% CO₂, the cloning efficiency was determined.

Flow cytometric analysis

During cell cycle assay, 3 × 10⁵ cells from each group were collected and combined with 70% cold ethanol overnight at 4°C. Cells were then washed with phosphate-buffered saline (PBS) three times and treated with 100 µg/mL RNase A for 0.5 h. After treating the cells with 50 µg/mL propidium iodide (PI) for 0.5 h in the dark, the DNA distribution was measured with a FACScan flow cytometer (BD Biosciences, San Jose, CA, USA). After staining the cells with the Annexin V-FITC/PI Apoptosis Detection Kit (BD Biosciences), the apoptosis rates of the samples were measured by a flow cytometer.

Wound healing assay

Cell migration was assessed using wound healing assay. After transfection, 1 × 10⁵ cells from each group were cultured to form a layer in 24-well plates. Subsequently, a 1-mm-wide symmetrical wound was created on the layer using a pipette. Cells were washed twice with Hanks’ balanced salt solution (HBSS) and then incubated for 24 h in a non-serum medium. The result of cells migration was photographed and the number was recorded at 0 and 24 h after drawing the wound. The number of migrated cells in these wells is counted as the sum of 10 random fields per well under a microscope (Abcam, Cambridge, MA, USA) at 100× magnification. Each cell count was performed in triplicate.

Transwell assay

Invasion analysis was conducted using an 8-µm Transwell chamber purchased from Corning Company in USA. A 200-µL non-serum cell suspension containing approximately 2 × 10⁴ cells was seeded into the upper Transwell chambers with 50 µL of Matrigel (1:4; BD Biosciences) 48 h after transfection. The lower chambers contained 500 µL of DMEM with 10% FBS. Cells were then cultured again for 24 h, and those that failed to pass through the filter membrane of the chambers were abandoned. Finally, the remaining cells were dyed with 0.1% crystal violet and counted from five random perspectives under a microscope. The counting was repeated three times for each group.

Statistical analysis

Data were analyzed using SPSS 21.0 software (Chicago, IL, USA). Enumeration data were analyzed using the chi-square test, and measurement data were expressed as mean ± standard deviation. Measurement data with a Gaussian distribution was analyzed using the t-test and one-way analysis of variance (ANOVA). p < 0.05 was considered statistically significant.

Results

Expressions of miR-1-3p and CCL2 in BC tissues and cells

First, the levels of miR-1-3p and CCL2 in BC tissues were detected using RT-PCR and western blot. There are no remarkable differences of miR-1-3p and CCL2 between the normal tissues and adjacent tissues (p > 0.05). BC tissues had a significantly lower expression of miR-1-3p and significantly higher expressions of CCL2 mRNA and protein than normal and adjacent tissues (p < 0.05, Figure 1(a)–(c)). Second, we detected the expressions of miR-1-3p and CCL2 in BC cell lines and normal cell line. We found that three BC cell lines (5637, T24, and UM-UC-3) had significantly lower miR-1-3p expression but higher expressions of CCL2 mRNA and protein (p < 0.05, Figure 1(d)–(f)). The expression levels of UM-UC-3 were considered outstanding; therefore, the cell line UM-UC-3 was chosen for further experiments.

Figure 1.

(a) Expression of miR-1-3p in different tissue samples, *p < 0.05. (b) Expression of CCL2 mRNA in different tissue samples, *p < 0.05. (c) Expression of CCL2 protein in different tissue samples. (d) Expression of miR-1-3p in different cell lines. *p < 0.05 compared with the normal group. (e) Expression of CCL2 mRNA in different cell lines. *p < 0.05 compared with the normal group. (f) Expression of CCL2 protein in different cell lines.

Targeting relationship between CCL2 and miR-1-3p

Computational analysis based on the TargetScan database predicts that CCL2 3′UTR has a binding site for miR-1-3p (Figure 2(a)). To confirm this targeting relationship, we conducted a luciferase reporter assay on the cell line UM-UC-3. As expected, the cells transfected with wild-type CCL2 and the miR-1-3p mimics had significantly lower luciferase intensity than the control group (p < 0.05). On the other hand, cells transfected with mutated CCL2 and miR-1-3p mimics showed no significant difference from the control group (p > 0.05, Figure 2(b)).

Figure 2.

(a) The target regions of miR-1-3p on the 3′UTR of CCL2; (b) luciferase intensity in UM-UC-3 cells transfected with wild type and mutated CCL2 3′UTR, *p < 0.05; and (c) western blot analysis shows that miR-1-3p downregulated CCL2 protein expression.

The results of the western blot analysis demonstrated that the miR-1-3p mimics could downregulate CCL2 protein expression significantly when compared with the control group and the NC group (Figure 2(c)).

Transfection of miR-1-3p mimics and CCL2 siRNA regulates cell proliferation, cell cycle, and apoptosis

We investigated the effects of miR-1-3p and CCL2 on the proliferation, cell cycle, and apoptosis of BC cells. After transfecting the miR-1-3p mimics and CCL2 siRNA, the MTT results demonstrated that cell viability was inhibited with statistical difference (p < 0.05, Figure 3(a)).

Figure 3.

(a) Transfection of miR-1-3p mimics and CCL2 siRNA affected cell viability in the UM-UC-3 cell line. (b and c) Transfection of miR-1-3p mimics and CCL2 siRNA affected cell proliferation in the UM-UC-3 cell line. *p < 0.05 compared with the control group. (d and e) Transfection of miR-1-3p mimics and CCL2 siRNA affected cell cycle in the UM-UC-3 cell line. *p < 0.05 compared with the control group. (f and g) Transfection of miR-1-3p mimics and CCL2 siRNA affected cell apoptosis in the UM-UC-3 cell line. *p < 0.05 compared with the control group.

The colony formation assay showed that the transfection of miR-1-3p mimics and CCL2 siRNA led to a significant decrease in the colony formation of UM-UC-3 cells (p < 0.05, Figure 3(b) and (c)).

By observing the cell cycle, we found that most cells transfected with miR-1-3p mimics and CCL2 siRNA were stagnated in the G0/G1 phase, the number of cells in the S phase were decreased, and the cell’s mitosis is suppressed. This means that very few cells are left in the G2/M phase (p < 0.05, Figure 3(d) and (e)).

Furthermore, the flow cytometric analysis results indicated that apoptosis was upregulated more in the mimics and siCCL2 groups than in the control and NC groups (p < 0.05, Figure 3(f) and (g)). In the above three assays, no obvious differences were observed between the miR-1-3p mimics group and the CCL2 siRNA group (p > 0.05, Figure 3).

Transfection of miR-1-3p mimics and CCL2 siRNA can regulate cell invasion and migration

To uncover the complicated functions of miR-1-3p and CCL2 in BC, the wound healing and Transwell assay were performed to determine the invasion and migration of the UM-UC-3 cell line after the transfection of miR-1-3p mimics and CCL2 siRNA. We found that exogenous miR-1-3p and the knockdown of CCL2 significantly inhibited the migration and invasion of UM-UC-3 cells (p < 0.05), while there was no remarkable difference between the miR-1-3p mimics group and the CCL2 siRNA group (p > 0.05, Figure 4).

Figure 4.

Transfection of miR-1-3p mimics and CCL2 siRNA affected cell migration and invasion in the UM-UC-3 cell line. *p < 0.05 compared with the control group.

Discussion

BC is the seventh most common cancer worldwide.^26,27 In China, it is the most common cancer among all of the urinary system tumors with a rising incidence every year.²⁸ Therefore, identifying its underlying mechanisms may aid us in novel therapeutic studies. In our research, we revealed that miR-1-3p was downregulated in BC cells and that by targeting CCL2 and decreasing its expression, a series of BC cell activities were inhibited. Our study may help develop important therapeutic strategies to combat BC.

MiR-1-3p is a small non-coding microRNA that regulates the translational repression or mRNA cleavage to modulate gene expression.^29,30 It has been proved that miR-1-3p regulates different gene expressions and therefore may regulate many human cancers such as prostate cancer,³¹ nasopharyngeal carcinoma,³² chordoma,³³ colorectal tumor,³⁴ and gastric cancer.³⁵ The relationship between BC and miR is in need of further research. Previous studies have shown that the expression of miR-1-3p is low in BC cells.^36–40 We confirmed this finding in our study. We found that both BC tissues and cell lines had lower expression of miR-1-3p than normal tissues and cell lines, respectively. We also identified that miR-1-3p is a tumor suppressor; however, the underlying mechanism of this suppression is still in need of further research. The results of TargetScan database identified several potential targets of miR-1-3p.

In this study, we focused on one of these potential targets (CCL2) of miR-1-3p to confirm that miR-1-3p influences the pathogenesis of BC. CCL2 is one chemokine which is a group of small secretory proteins.⁴¹ On the surface of endothelial cells, CCL2 reacts with its receptor CCR2, which therefore promotes the vessel sprout formation and angiogenesis.^42,43 The chemokine CCL2 has been identified to be an important characteristic substance in many human cancers such as breast cancer,⁴⁴ ovarian cancer,⁴⁵ and lung cancer.⁴⁶ According to previous researches, the polymorphisms of CCL2 and its receptors are related to the transitional bladder cell carcinoma.^47,48 A few studies have also reported that CCL2 is a key contributor in mediating the migration and invasion of BC cells in the signaling pathways. However, this is under further investigation.^22,49 Our research helps to confirm this finding as we demonstrated that CCL2 is highly expressed in BC tissues and cell lines. Furthermore, we also indicated that the low expression of CCL2 could inhibit the metastasis and the proliferation of BC cells.

Although we have already predicted the relationship between CCL2 and miR-1-3p through the TargetScan database, the relationship is still in need of confirmation. We further designed a set of experiments to test this relationship. When we increased the expression of miR-1-3p, a lower expression of CCL2 was observed. The luciferase reporter assay also confirmed a negatively modulating effect of miR-1-3p on CCL2. It is the first time that this relationship has been reported, and it may provide a potential new therapy against BC.

miR exercises its function by pairing to 3′UTR at the base of target genes.⁵⁰ According to the previous reports, many oncogenes have been identified to be related to metastasis and the proliferation of BC cells, c-erb-B2,⁵¹ KLF5,⁵² CD44,⁵³ and so on. In our study, we predicted that CCL2 is one of these oncogenes and is a target gene of miR-1-3p. Rao et al.⁴⁹ demonstrated that CCL2 is an important node to the signaling pathway that modulates BC metastasis. According to their research, Erβ (estrogen receptor beta)/CCL2/CCR2 (C-C chemokine receptor-2)/EMT (epithelial–mesenchymal transition)/MMP9 (matrix metallopeptidase-9) forms a signaling pathway which primarily modulates metastasis. The results of our experiments are in line with their research findings. CCL2 does play an important role in the metastasis of BC, which is consistent with the results of a study that Chiu et al.²² carried out in 2012. Furthermore, we also revealed that CCL2 has a great influence on the behavior of proliferation. MiR-1-3p mimics display similar effects to CCL2 siRNA on the proliferation, migration, and invasion of BC cells.

However, further research with the aim of exploring and fully understanding the mechanism behind how miR-1-3p suppresses BC is needed. From the results of the database, we can see that CCL2 is not the only target gene of miR-1-3p. Therefore, the research on the relationship between miR-1-3p and other genes is also required for a complete understanding of its effects. Moreover, miR-1-3p is not the only miR that targets CCL2; as such, research on the relationship of other miR and CCL2 may also be meaningful to understand how to constrain BC cells.

In conclusion, we confirmed that the overexpression of miR-1-3p could inhibit BC cells by targeting CCL2. Most significantly, we proved for the first time that there is a direct target relationship between miR-1-3p and CCL2. Furthermore, we found that miR-1-3p could restrain the activity of BC cells by downregulating the expression of CCL2 (suggesting a negatively modulating relationship). Our research may provide a new treatment strategy for BC.

Footnotes

Acknowledgements

Weiwei Wang and Fujun Shen are the first authors.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work is supported by 2015 Postgraduate Education Innovation Program (NXYC201511).

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