Abstract
Introduction
Lung cancer is one of the most commonly seen tumors in the world with abnormal proliferation of cells in the lung tissues.1,2 Small cell lung cancer and non–small cell lung cancer (NSCLC) are the two main types of lung cancers.3,4 Surgery is the primary treatment for patients with localized disease and without spreading to the lymph nodes. Besides, lobectomy, radiation therapy, chemotherapy, or combination therapy are also the most commonly used therapy in lung cancer patients.5,6 However, none of them can completely eradicate the malignant lung tumors and inhibit the progression of lung cancers. The possibility of recurrence and metastasis of lung cancer still existed. Thus, the new treatment and novel biomarker for diagnosis and treatment of human lung cancers are the current hotspots.
MicroRNA (miRNA) is a cluster of newly discovered non-encoding RNA molecules with 21–23 nucleotides long.7,8 They are transcribed by RNA polymerase II and play important roles in development of various human diseases, such as the tumorigenesis of human cancers.9,10 A large number of studies have shown that aberrant miRNA expression is casual in various human cancers. 11 The dysregulated miRNAs act as tumor suppressors or onco-miRNAs in the cancer progression and possess a promising potential to treat cancers in clinical therapy. For example, the first miRNA-targeted drug, miravirsen or SPC3649, as the miR-122-specific inhibitor, has been used to treat hepatitis C virus (HCV) infection in phase II clinical trials. 12 Now, enormous miRNAs were identified and found to be involved in the progression and metastasis of lung cancers. Chen et al. 13 reported that miR-126 and miR-133b were downregulated in NSCLC and contributed to the progression and metastasis of NSCLC. MiR-585 level was decreased in lung cancer tissues and NSCLC cell lines. Overexpression of miR-585 suppressed cell viability, migration, and invasion in vitro, as well as decreased the growth of xenografted tumor in vivo. The miR-585 played the inhibitory effects on tumor growth by targeting the 3′ untranslated region (UTR) of hSMG-1 gene. 14 Moreover, miR-376c, 15 miR-200c, 16 miR-26b, 17 miR-338-3p, 18 miR-98, 19 and miR-455 20 suppressed the invasion and metastasis of lung cancer cells by targeting their downstream regulators. Thus, it is promising to identify and clarify the efficacious miRNA in lung cancer therapy.
MiR-557 was a new miRNA to be found recently, and its function in cancer progression was not clearly clarified till now. It was first screened in gastric cancer tissues by microarray data from the Gene Expression Omnibus (GEO) database. 21 In Ewing’s sarcoma (ES), miR-557 showed recurrently altered expression and was predicted to regulate ES-associated genes in insulin-like growth factor 1 (IGF1) pathway. 22 However, there was no statistical difference on miR-557 level in the serum of patients with nephrotic syndrome subtypes and healthy controls. 23 To date, the function of miR-557 and the possible regulatory mechanism have not been studied in lung cancer cells. At present, we identified the role of miR-557 in the progression of human lung cancers and investigated the possible regulatory mechanism in lung cancer cells. All the data could provide new clues for the clinical diagnosis and therapy for human lung cancer patients.
Materials and methods
Specimens
In total, eight pairs of clinical specimens (lung cancer tissues and paracancer tissues) were obtained from The First Affiliated Hospital and College of Clinical Medicine of Henan University of Science and Technology. There were three female and five male patients (age: 48–61 years old). The specimens were quickly frozen in liquid N2 and kept at −80°C in a refrigerator. All the patients signed the relevant contracts prior to the experiment. The studies carried out on humans were in compliance with the Helsinki Declaration and approved by the Ethics Committee of The First Affiliated Hospital and College of Clinical Medicine of Henan University of Science and Technology.
Cell lines and agents
The normal human epithelial cells (BEAS-2B cells; Cat. No. GF083) and human NSCLC cells (HCC-827; Cat. No. GF204) were purchased from Shanghai Gefan Biotechnology, Co., Ltd (Shanghai, China). BEAS-2B cells were derived from normal human bronchial epithelium after autopsy of non-cancerous individuals. The lung squamous cell carcinoma SK-MES-1, human adenocarcinoma cell A549, and NCI-H460 were cultured and kept in our laboratory. The cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) at 37°C in a humidified 5% CO2 atmosphere. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) agent was obtained from Sigma-Aldrich (St. Louis, MO, USA). Hsa-miR-557 miRNA mimic (Cat. No. mch03075) and miRNA mimic negative control (Cat. No. MCH00000) were obtained from Applied Biological Materials Inc. (Richmond, Canada).
MTT assay
Lung cancer cells (A549 and NCI-H460) were transfected with miR-557 mimics and negative control mimics for 24, 48, and 72 h, respectively. MTT assay was performed to detect the cell viability of lung cancer cells. The cells transfected with negative control mimics were used as negative controls. Then, 4 h before test, a volume of 10 µL of MTT (store concentration of 5 mg/mL) was added into the cultured medium. The purple crystal was dissolved by 100 µL of dimethyl sulfoxide (DMSO). Finally, the absorbance of each well in 96-well plate was read at 490 nm.
RNA extraction and quantitative real-time polymerase chain reaction analysis
The complementary DNA (cDNA) synthesis kit for miRNA (Cat. No. HP100041) and miR-557 (human) qSTAR miRNA primer pairs (HP300471) were obtained from OriGene Technologies, Inc. (Beijing, China). Total RNA was extracted from 2 × 106 lung cancer cells using the mirVANA™ miRNA Isolation Kit (Cat. No. AM1560; Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. Total RNA (10 ng) was reverse transcribed into cDNA using Taqman MicroRNA Assay (Life Technologies, San Diego, CA, USA) and cDNA synthesis kit for miRNA (Cat. No. HP100041; OriGene Technologies) according to the manufacturer’s instructions. Real-time polymerase chain reaction (RT-PCR) was performed with miR-557 (human) qSTAR miRNA primer pairs (HP300471; OriGene Technologies) using a Cfx96 device (Bio-Rad, Hercules, CA, USA). The reaction conditions were as follows: activation at 50°C for 2 min and pre-soak at 95°C for 10 min; followed by 42 cycles of denaturation at 95°C for 15 s, annealing at 55°C for 10 s, and extension at 72°C for 30 sec. ΔCq values were calculated with the single threshold method (Bio-Rad CFX Manager Software 2.1; Hercules, CA, USA). U6 small nuclear 2 (RNU6B) was used as an internal reference.
Fluorescence-activated cell sorting assay
The lung cancer cells were plated onto six-well plate. After 8 h, the cells were transfected with miR-557 mimics and negative control mimics for 48 h. Cells were then harvested and stained with Annexin V–fluorescein isothiocyanate (FITC) and propidium iodide (PI) apoptosis detection kit (Cat. No. 40302ES20; Yeasen, USA) according to the protocol. The cells were stained with Annexin V–FITC and PI for 15 min and then analyzed on a cytofluorimeter by Coulter Epics XL equipped with system II software (Beckman Coulter, Miami, FL, USA).
In the other experiment, the lung cancer cells were washed with cold phosphate-buffered saline (PBS) and resuspended at a concentration of 3 × 106/mL for analysis. Next, 2°µL of a PI stock solution (500°µg/mL) was added to each sample and mixed thoroughly. The samples were kept at 4°C in dark for 5 min and analyzed on a Coulter a XL equipped with system II software (Beckman Coulter).
Western blotting analysis
A549 and NCI-H460 cells were treated with miR-557 mimics and negative control mimics for 48 h. The levels of lymphocyte enhancement factor 1 (LEF1) and TCF4 were determined by western blotting as described.24–26 The primary antibodies used were as follows: Anti-LEF1 antibody [EP2030Y] (ab53293), a rabbit monoclonal [EP2030Y] to LEF1 and Anti-TCF4 antibody (ab185736), a rabbit polyclonal to TCF4. Both of them were obtained from Abcam (Cambridge, MA, USA). β-actin was used as an internal reference gene. β-Actin Antibody (C4): sc-47778 was a mouse monoclonal IgG1 and was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Migration assay
The migration ability of lung cancer cells was detected by transwell assay. Briefly, A549 cells were transfected with miR-557 mimics and negative control mimics for 24 h. The cells transfected with miR-557 mimics or negative control mimics were plated in the upper chambers. Then, 600°µL of medium containing 20% FBS was added to the bottom chambers as the chemotactic factor. After 48 h, the migrated cells were fixed using methyl alcohol, stained using 0.1% crystal violet, and visualized (original magnification: 200×) using a light microscope.
Statistical analysis
All the data were analyzed by SPSS 20.0 (IBM SPSS, Armonk, NY, USA). All experiments were carried out in triplicates independently. The results were shown as mean ± standard error. p < 0.05 was considered to be statistically significant.
Results
MiR-557 expression is downregulated in lung cancer specimens and lung cancer cell lines
In order to clarify the effects of miR-557 in the progression of human lung cancers, RT-PCR was performed to detect the levels of miR-557 in eight pairs of clinical specimens of lung cancer tissues and the paired paracancer tissues. In Figure 1(a), miR-557 was significantly downregulated in human lung cancer tissues compared with the paired paracancer tissues (*p < 0.01). In addition, miR-557 levels were determined by RT-PCR in human lung cancer cell lines (SK-MES-1, A549, HCC-827, and NCI-H460) and normal human bronchial epithelium (BEAS-2B). As shown in Figure 1(b), miR-557 was decreased obviously in lung cancer cell lines compared with the normal bronchial epithelium cells (*p < 0.05, **p < 0.01). The results revealed that lower levels of miR-557 might promote the carcinogenesis of lung cancers.
MiR-557 expression is downregulated in lung cancer specimens and lung cancer cell lines. (a) The levels of miR-557 were determined by real-time PCR in eight pairs of lung cancer tissues and paired paracancer tissues (*p < 0.05). The internal control was set using U6snRNA. (b) The expression level of miR-557 in lung cancer cell line was shown in histogram (*p < 0.05 and **p < 0.01, compared with that in BEAS-2B cells).
MiR-557 suppresses the proliferation of lung cancer cells
In order to clarify the effects of miR-557 on the proliferation of human lung cancer cells, the lung cancer cells A549 and NCI-H460 cells were transfected with miR-557 mimics and negative control mimics for indicated time, and MTT assay was used to determine the cell viability of the cells in each group. As shown in Figure 2(a) and (b), A549 and NCI-H460 cells were transfected with miR-557 mimics and negative control mimics for 24, 48, and 72 h, respectively, and the results demonstrated that higher levels of miR-557 significantly decreased the cell viability of A549 and NCI-H460 (*p < 0.05 and **p < 0.01, compared with negative control mimic–transfected cells).
MiR-557 suppresses the proliferation and promotes cell death of lung cancer cells. (a) The lung cancer cells (A549 and NCI-H460) were transfected with miR-557 mimics and negative control mimics for 48 h. The miR-557 level was determined by real-time PCR assay. Relative expression of miR-557 was shown in histogram in A549 and NCI-H460 cells (**p < 0.01, compared with negative control mimic–transfected cells). (b) A549 and NCI-H460 cells were transfected with miR-557 mimics and negative control mimics for 24, 48, and 72 h, respectively. The cell viability was determined by MTT assay, and the results were shown in histogram (*p < 0.05 and **p < 0.01, compared with negative control mimic–transfected A549 cells and NCI-H460 cells). (c) A549 cells were transfected with miR-557 mimics and negative control mimics for 72 h. The cells were stained with PI and analyzed by FACS as described in Material and methods section. (d) The rate of PI-positive cells was shown in histogram (**p < 0.01, compared with negative control mimic–transfected A549 cells or NCI-H460 cells.
MiR-557 mimics promote cell death of A549 and NCI-H460 cells
The lung cancer cells were transfected with miR-557 mimics and negative control mimics for 72 h; necrosis rate of lung cancer cells was determined by fluorescence-activated cell sorting (FACS) with PI staining. As PI could not cross the membrane of live cells, it was used to measure the cell rate of dead cells. As shown in Figure 2(c) and (d), the rate of PI-positive A549 cells and NCI-H460 cells in miR-557 mimic–transfected group was significantly higher than that in negative control mimic–transfected group (**p < 0.01). The results revealed that transfection with miR-557 mimics promoted cell death of lung cancer cells.
Transfection with miR-557 mimics does not affect cell apoptosis and cell cycle distribution of A549 and H460 cells
In order to detect whether miR-557 was involved in cell apoptosis in lung cancer cells, A549 and NCI-H460 cells were transfected with miR-557 mimics and negative control mimics combined with 10 ng/mL of actinomycin D for 48 h. Actinomycin D was an apoptosis inducer and used to induce cell apoptosis in the study. Annexin V–FITC/PI dual staining was used to determine cell apoptosis rate and necrosis rate in A549 cells (Figure 3(a)). As shown in Figure 3(b), the necrosis rate of A549 and NCI-H460 in miR-557 mimic–transfected group was significantly increased than the negative control mimic–transfected group (**p < 0.01), but there was no obvious difference in the apoptosis rates between miR-557 mimic–transfected group and control group (p > 0.05). Moreover, cell cycle distribution was analyzed by FACS with PI staining in lung cancer cells A549 and NCI-H460. As shown in Figure 3(c), the results showed that transfection of miR-557 mimics had no obvious variation on cell cycle distribution of A549 and H460 cells. All the data suggested that transfection with miR-557 increased cell death, but there was no obvious effect on the cell apoptosis in A549 cells.
Transfection with miR-557 mimics does not affect cell apoptosis of A549 cells. A549 cells were transfected with miR-557 mimics and negative control mimics and cultured for 48 h with actinomycin D at the concentration of 10 ng/mL. (a) Cell apoptosis was determined by FACS assay with Annexin V–FITC/PI dual staining method. (b) The apoptosis rate and necrosis rate were shown in histogram (**p < 0.01, compared with negative control mimic–transfected cells). (c) Cell cycle distribution: Cell cycle was analyzed by FACS with PI staining in lung cancer cells A549 and NCI-H460. The cell cycle distribution was shown in histogram.
Overexpression of miR-557 mimics inhibits cell invasion of A549 and NCI-H460
In order to explore the function of miR-557 in cell migration of lung cancer cells, transwell assay was performed in A549 and NCI-H460 cells. The A549 and NCI-H460 cells were transfected with miR-557 mimics and negative control mimics for 48 h. As shown in Figure 4, the image was observed at 48 h, and the results showed that transfection with miR-557 mimics significantly decreased lung cancer cell invasive capability (**p < 0.01, compared with negative control mimic–transfected cells). There was no statistical difference between the negative control mimic–transfected lung cancer cells and the untreated cells. The result revealed that miR-557 had an important role in inhibiting in vitro invasive ability of lung cancer cells.
Overexpression of miR-557 mimics inhibits cell migration and invasion of A549 and NCI-H460. (a) Migration ability was determined by transwell assay. The lung cancer cells (A549 cells and NCI-H460 cells) were transfected with miR-557 mimics and negative control mimics for 48 h. The image was captured with 200× magnification. (b) The relative cell migration rate was shown in histogram (**p < 0.01, compared with negative control mimic–transfected cells).
Hsa-miR-557 negatively regulates the expression of LEF1
LEF1 gene is expressed in multiple tissues and plays important roles at different stages of embryonic development, as well as mediating tumor occurrence. In this study, we used TargetScan to predict the target gene of hsa-miR-557; as shown in Figure 5(a), TargetScan analysis results demonstrated that hsa-miR-557 probably binds to the 3′UTR of LEF1 gene suggesting hsa-miR-557 might be involved in the tumorigenesis or metastasis of human cancers by regulating LEF1.
Hsa-miR-557 negatively regulates the expression of LEF1. (a) The possible target of hsa-miR-557 was predicted by TargetScan. The position 453–460 of LEF1 3′UTR in humans had binding sites for hsa-miR-557. (b) A549 cells were treated with miR-557 mimics and negative control mimics for 48 h. The expression of LEF1 and TCF4 was determined by western blotting analysis. The gray value of LEF1 was shown in histogram (**p < 0.01, compared with negative control mimic–transfected cells).
Next, we tested whether LEF1 expression was regulated by miR-557 in A549 and NCI-H460 cells. Briefly, A549 and NCI-H460 cells were transfected with miR-557 mimics and negative control mimics for 48 h, and the levels of LEF1/TCF4 were tested by western blotting. As shown in Figure 5(b), overexpression of miR-557 mimics significantly decreased the expression level of LEF1 in A549 and NCI-H460 cells (**p < 0.01, compared with negative control mimic–transfected group), but TCF4 expression had no statistical difference in miR-557 mimic–transfected cells and negative control mimic–transfected cells (p > 0.05). All the data suggested that hsa-miR-557 suppressed the proliferation or invasion of lung cancer cells probably by negatively regulating the LEF1 factor.
Discussion
MiR-557 is a novel miRNA, and the effects of miR-557 are not clarified in the tumorigenesis of human lung cancers. In order to identify the role of miR-557 in the progression of human lung cancers, we first detected the expression of miR-557 in eight pairs of lung cancer specimens and the paracancer tissues. The RT-PCR assay results obviously showed that miR-557 expression was lower in lung cancer tissues than that in the paired paracancer tissues. We need to perform further study and test more clinical specimens to identify whether miR-557 would be used as the useful biomarker for screening, monitoring, or predicting prognosis in lung cancer patients.
Then, the anti-tumor effects of miR-557 were studied, as well as the potential mechanism of miR-557 in human lung cancer cell line, A549 and NCI-H460. First, the effects of miR-557 on cell proliferation, cell apoptosis, and invasion were explored. Here, we used two kinds of methods to detect the cell death of lung cancer cells, including MTT assay and FACS analysis with PI staining. The results were consistent and both results revealed that miR-557 mimics significantly inhibited lung cancer cell proliferation. However, there was no significant difference on cell apoptosis rate and cell cycle distribution between the cells transfected with miR-557 mimics and negative control mimics. The data provide a clue that the regulatory mechanism of miR-557-induced cell death was probably not due to cell apopotosis.
Moreover, transwell assay was performed to detect the invasion ability of A549 and NCI-H460 cells transfected with miR-557 mimics and negative control mimics. The results demonstrated that overexpression of miR-557 mimics obviously inhibited cell migration of lung cancer cells. In order to investigate the exact molecular mechanism of miR-557 in the migration and invasion of lung cancers, we used TargetScan to analyze the target gene of miR-557 in humans. Interestingly, the results by TargetScan analysis demonstrated that miR-557 might bind with 3′UTR of LEF1. In order further to know whether miR-557 directly binds with the 3′UTR of LEF1, we will test it by using the luciferase reporter gene assay later. It has been reported that LEF1 played an important role in the migration of lung cancer cells. As we know, LEF1 is a key transcription factor to regulate the tumorigenesis of various tumors. Numerous matrix metalloproteinase (MMP) family members are downstream effectors of the Wnt/LEF1 signaling pathway. It has been reported that miR-218 inhibited the invasive activity of glioblastoma multiforme (GBM) cells by targeting LEF1 and blocked the invasive axis, miR-218-LEF1-MMPs. 27 Moreover, LEF1 also altered the invasion ability of cancer cells by regulating the expression of miRNAs. For example, LEF1 knockdown decreased miR-181a expression via binding to the promoter regions of hsa-miR-181a leading to inhibition of epithelial–mesenchymal transition (EMT), migration, and invasion. Thus, LEF1-miR-181a-EMT axis played an important role in the regulation of androgen-independent prostate cancer migration and invasion. 28 In this study, we found that miR-557 downregulated the expression of LEF1 in lung cancer cells. Next, to know whether the decreased expression of LEF1 contributed to the inhibition of cell migration and invasion of lung cancer cells, the clear molecular mechanism needs to be further explored which will be helpful for developing potential clinical strategies.
In conclusion, miR-557 demonstrated anti-tumor effects in the proliferation and invasion of human lung cancer cells. Overexpression of miR-557 promoted cell death of lung cancer cells without altering the cell apoptosis and cell cycle distribution. Higher expression of miR-557 inhibited cell migration of lung cancer cells probably by negatively regulating the expression of LEF1 in human lung cancer cells. Thus, miR-557 worked as a tumor suppressor in the proliferation and invasion of lung cancer cells.