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Introduction

Lung cancer is a notable cause of mortality and is the 2nd most common type of cancer worldwide, of which non-small cell lung cancer (NSCLC) accounts for 80–85% of cases (1,2). The majority of patients with NSCLC are diagnosed in an advanced disease stage; therefore, the disease is associated with a poor 5-year survival rate (3). Due to advances in molecular biology, treatments for adenocarcinoma have improved; treatments for lung cancer, however, remain unsatisfactory, and the incidence and mortality rate of patients with lung cancer remain high (4,5).

MicroRNAs (miRNAs/miRs) are small, non-coding RNA molecules which serve important roles as regulatory factors in carcinogenesis (6,7). miRNAs have been reported to be involved in multiple types of tumor, including lung cancer. The discovery of miRNAs may provide a novel and powerful tool for researching the mechanism, diagnosis and treatment of lung cancer. Previous research has characterized miRNAs as a group of novel therapeutic small molecules involved in the treatment and regulation of lung cancer. Abnormal expression of miRNAs in plasma has been observed in some patients with malignant tumors, such as miR-30a, which was demonstrated to be overexpressed in NSCLC cells (8). Chen et al (9) reported that miR-206 is underexpressed in lung cancers and may be a potential target for therapy by inhibiting epithelial-mesenchymal transition and angiogenesis in lung cancer. With the aim of investigating the potential role of miR-95 in the treatment of NSCLC, Ma et al (10) and Chen et al (11) investigated the expression level of miR-95 and observed it to be overexpressed in recurrent NSCLC, and demonstrated that miR-95a is a potential therapeutic target for the treatment of NSCLC.

Metastasis is recognized as a frequent cause of mortality in patients with NSCLC. Previous studies have demonstrated the roles of miR-10b and miR-145 in the invasive and metastatic capabilities of lung cancer cells, and that miR-10b upregulated the migration and invasion of lung cancer cells, while miR-145 suppressed migration and invasion (12–15). These previous results provide a potential approach for developing miRNA-based therapeutic strategies for the treatment of NSCLC. In a correlation study of miR-21 in lung cancer cells, miR-21 was investigated as a potential serum biomarker, and diagnostic and prognostic indicator for NSCLC (16–18). However, the molecular mechanism underlying the role of miR-21 in lung cancer remains to be elucidated.

The objective of the present study was to investigate the association between miR-21 expression, cell viability and apoptosis in lung cancer. The results of the present study demonstrated that miR-21 was able to increase the viability of A549 cells by inhibiting cellular apoptosis. In addition, the signaling pathway of miR-21 in the regulation of lung cancer cell lines was investigated, and the results demonstrated that miR-21 inhibited cellular apoptosis by modulating the activation of the phosphatidylinositol 3-kinase (PI3K)/Rac-α serine/threonine protein kinase (Akt) pathway in A549 cells. Correspondingly, inhibition of Akt using MK-2206 decreased the rate of apoptosis in miR-21 knockdown A549 cells. The results of the present study may provide a theoretical basis for, and novel insights into, the treatment of lung cancer.

Materials and methods

Cell culture and transfection

A549 cells were purchased from the American Type Culture Collection (Manassas, VA, USA). Cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C in a humidified atmosphere with 5% CO2. The cells were transfected with miR-21 (Lipo miR-21 group), small interfering (si)RNA against miR-21 (5′-UCAACAUCAGUCUGAUAAGCUA-3′) or mismatch siRNA as a negative control (5′-UCUUCAUGAGUCAGAUUACCUA-3′). All transfections were performed by using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Additionally, after transfection for 48 h, certain cells that were transfected with miR-21 siRNA were treated with the Akt inhibitor MK-2206 at room temperature for 24 h (20 µM; Selleck Chemicals, Houston, TX, USA).

Cell viability assay

For transfection, cells were cultured on 12-well plates and seeded at a density of 5×104 cells/well for 48 h at 37°C. The cells were harvested using trypsin, re-suspended in 3 ml culture medium, and counted with a hemocytometer. Cell samples were collected at 0, 24 and 48 h after transfection for further analysis. For the MTT assays, transfected cells at a density of 5×103 cells/well were seeded onto 96-well culture plates. After 24 h incubation at 37°C, cell viability was assayed by adding 10% MTT (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) to 0.2 ml culture medium and incubating at 37°C for 3 h. Following removal of the medium, formazan crystals were dissolved with 100 µl dimethyl sulfoxide (Sigma-Aldrich; Merck KGaA) for 10 min at room temperature, and the optical density was measured at 590 nm with a Multiskan EX (Thermo Fisher Scientific, Inc.). The assay was repeated 3 times (19).

Cell cycle analysis using flow cytometry

Cells were harvested by trypsinization and washed with PBS. The cells were fixed with 100% ice-cold methanol at −20°C overnight. The cells were subsequently incubated for 30 min in 50 µg/ml propidium iodide with 1 mg/ml RNase. The apoptotic cells were observed at 450 nm excitation wavelength by using a FACScan flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). Data were analyzed using CellQuest 3.1f software (BD Biosciences). Each condition was repeated ≥3 times (20).

RNA extraction and the reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

Total RNA was extracted using TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.) and reverse transcribed using an RT kit (Toyobo Co., Ltd., Osaka, Japan). miRNAs were extracted using the All-in-One miRNA RT kit (GeneCopoeia, Inc., Rockville, MD, USA). cDNA was subjected to qPCR using SYBR Premix Ex Taq (Takara Biotechnology Co., Ltd., Dalian, Japan) using StepOnePlus (Invitrogen; Thermo Fisher Scientific, Inc.). qPCR conditions were as follows: Initial denaturation at 95°C for 15 sec; 30 cycles of denaturation at 95°C for 30 sec, annealing at 61°C for 5 sec and extension at 72°C for 15 sec; final extension at 72°C for 10 min. miR-21 expression was normalized to U6 using the 2−ΔΔCq method (21). Primers for miR-21 and U6 were purchased from GeneCopoeia, Inc. U6 primers: forward, 5′-CTTCGGCAGCACATATACT-3′; and reverse, 5′-AAAATATGGAACGCTTCACG-3′. miR-21 primers: forward, 5′-ACGTTGTGTAGCTTATCAGACTG-3′; and reverse, 5′-AATGGTTGTTCTCCACACTCTC-3′.

Western blot analysis

Cells were washed with PBS and lysed in a lysis buffer [pH 7.4; 20 mM Tris-HCl, 0.1 mM EDTA, 0.1% SDS, 20 mM NaF, 150 mM NaCl, 0.1% Triton X-100, 1% NP-40, 1 mM Na3VO4 and 1X protease inhibitor (Roche Diagnostics, Basel, Switzerland)]. The proteins were quantified using the BCA Protein assay kit (Pierce; Thermo Fisher Scientific, Inc.). Protein samples (30 µg per lane) were separated using 10–12% SDS-PAGE and transferred to a nitrocellulose membrane (Whatman; GE Healthcare Life Sciences, Little Chalfont, UK) following boiling for 10 min in SDS sample buffer (Sigma-Aldrich; Merck KGaA). Following blocking with 5% skimmed milk in TBS with Tween-20 (TBS-T) for 1 h at room temperature, the membranes were incubated with primary antibodies against phosphorylated (p)-Akt (cat. no. 4060; 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA), Akt (cat. no. 9272; 1:1,000; Cell Signaling Technology, Inc.), B-cell lymphoma 2 (Bcl-2; cat. no. ab32124; 1:1,000; Abcam, Cambridge, UK), Bcl-2-associated X (BAX; cat. no. ab32503; 1:1,000; Abcam), procaspase-3 (cat. no. ab32150; 1:1,000; Abcam), active caspase-3 (cat. no. ab2302; 1:1,000; Abcam) and actin (cat. no. ab8227; 1:1,000; Abcam) overnight at 4°C. Membranes were washed with TBS-T and incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (cat. no. ab205718; 1:5,000; Abcam) for 2 h at room temperature. Protein bands were visualized using the WEST-ZOL-plus Western Blot Detection System (22).

Statistical analysis

Data are presented as the mean ± standard deviation. All statistical analyses were performed using GraphPad Prism 5.0 software (GraphPad Software, Inc., La Jolla, CA, USA). P-values were calculated using one-way analysis of variance. P<0.05 was considered to indicate a statistically significant difference.

Results

Overexpression of miR-21 increases cell viability in A549 cells

The mRNA expression of miR-21 is presented in Fig. 1A. The results demonstrated that miR-21 expression was significantly upregulated by miR-21 overexpression, but was downregulated by miR-21 suppression, compared with their corresponding controls (P<0.05). An MTT assay was used to determine the viability of A549 cells following 0, 24 and 48 h of transfection with miR-21, mismatch siRNA or miR-21 siRNA. The results presented in Fig. 1B demonstrated that overexpression of miR-21 significantly increased cell viability in A549 cells. However, the result was reversed by suppression of miR-21.

Figure 1. Cell viability of different transfected groups. (A) Expression of miR-21 in the control, miR-21 overexpressing, mismatch siRNA and miR-21 siRNA groups. miR-21 was overexpressed by miR-21 transfection and effectively inhibited by miR-21 siRNA. (B) Cell viability results from the MTT assay of all groups. Error bars represent the mean ± standard deviation. *P<0.05 or **P<0.01 vs. control; #P<0.05 or ##P<0.01 vs. mismatch siRNA. miR, microRNA; siRNA, small interfering RNA.

Overexpression of miR-21 promotes cell proliferation by inhibiting cellular apoptosis

The results of the flow cytometry demonstrated that the apoptotic rates of the A549 cells in the control, miR-21, mismatch siRNA and miR-21 siRNA groups were 9, 5, 8 and 28%, respectively (Fig. 2). The results of the present study demonstrated that the apoptosis rate of A549 cells in the miR-21-treated group significantly decreased compared with the control (P<0.05). However, suppression of miR-21 significantly increased cell apoptosis in A549 cells compared with the mismatch siRNA group (P<0.05).

Figure 2. Cellular apoptosis of different transfected groups. Cells overexpressing miR-21 exhibited a lower apoptotic rate. Inhibition of miR-21 was associated with increased apoptotic rate. Error bars represent the mean ± standard deviation. *P<0.05 vs. control; #P<0.05 vs. mismatch siRNA. miR, microRNA; siRNA, small interfering RNA; FL, fluorescence; LIN, lineage.

miR-21 inhibits apoptosis by modulating the activation of the PI3K/Akt signaling pathway in A549 cells

The apoptosis inhibition mechanism of miR-21 in A549 cells was examined by analyzing the protein expression of associated factors. The western blotting results for p-Akt, Akt, Bcl-2, BAX, procaspase-3, active caspase-3 and actin are presented in Fig. 3. The results demonstrated that overexpression of miR-21 upregulated p-Akt and Bcl-2 expression, and downregulated the expression of BAX and procapase-3, compared with the control group. Suppression of miR-21 demonstrated the opposite effect on these factors. There were no obvious changes in the expression of Akt and active caspase-3 following miR-21 overexpression or suppression. The results of the present study demonstrated that miR-21 inhibited apoptosis in A549 cells by modulating the activation of the PI3K/Akt signaling pathway.

Figure 3. Phosphatidylinositol 3-kinase/Akt signaling pathway is involved in miR-21-mediated inhibition of cellular apoptosis. Western blotting results of apoptosis-associated proteins. p-, phosphorylated-; Akt, Rac-α serine/threonine protein kinase; Bcl-2, B-cell lymphoma 2; BAX, Bcl-2-associated X; miR, microRNA; siRNA, small interfering RNA.

Inhibition of Akt decreases the rate of apoptosis in A549 cells

The expression of Akt was inhibited and the apoptosis rate of the A549 cells was measured. The miR-21 siRNA + Akt inhibitor cells exhibited a decreased apoptotic rate compared with the miR-21 siRNA group, as presented in Fig. 4. The results demonstrated that the apoptotic rates of the A549 cells in the control, miR-21, mismatch siRNA, miR-21 siRNA and miR-21 siRNA + Akt inhibitor groups were 9, 4.3, 8, 28 and 9.3%, respectively. miR-21 overexpression significantly inhibited cell apoptosis compared with the control group, whereas miR-21 suppression promoted apoptosis as apoptosis was significantly increased compared with the mismatch siRNA group. However, the promoting effect of miR-21 suppression on cell apoptosis was abolished by treatment with an Akt inhibitor, as apoptosis was significantly reduced in the miR-21 siRNA + Akt inhibitor group compared with the miR-21 siRNA-only group. These results indicated that inhibition of Akt decreased the rate of apoptosis in A549 cells when miR-21 was suppressed.

Figure 4. Apoptotic index of transfected cells. miR-21 siRNA + Akt inhibitor cells exhibited decreased apoptosis compared with the miR-21 siRNA group. Error bars represent the mean ± standard deviation. *P<0.05 and #P<0.05, as indicated. miR, microRNA; siRNA, small interfering RNA; Akt, Rac-α serine/threonine protein kinase; FL, fluorescence; LIN, lineage.

Discussion

NSCLC is one of the most common types of malignant tumor worldwide (23). It is associated with the highest incidence of morbidity and mortality, and smoking is the primary risk factor (1). A lack of effective detection methods has led to the ineffectiveness of conventional therapy. The prognosis of NSCLC is poor, due to insufficient mechanistic understanding.

As one of the most important regulators of the pathogenesis of human disease through the regulation of gene expression, miRNAs has been reported to serve roles in the progression of human lung cancer, including miR-196a, miR-200c and miR-204 (24–26). miR-21 is one of the miRNAs reported to be associated with apoptosis in human NSCLC cells (17,18). However, more research into the potential role of miR-21 is required. In order to investigate the molecular pathogenesis of NSCLC, and to verify whether miR-21 may be a molecular diagnostic and prognostic marker in NSCLC, the present study sought to investigate the molecular mechanisms in detail.

In the present study, miR-21 was overexpressed in A549 cells and cell viability was assayed following transfection with miR-21, mismatch siRNA or miR-21 siRNA. The results obtained in the MTT assay demonstrated that miR-21 was able to increase the viability of A549 cells. The apoptotic rate of A549 cells was investigated, and it was demonstrated that miR-21 increased viability by inhibiting cellular apoptosis.

The activation of apoptotic pathway serves an important role in biological development and tissue homeostasis (27). Proteins in the Bcl-2 family, including Bcl-binding component 3, BAX and Bcl-2 homologous antagonist/killer, are downregulated by numerous anti-apoptotic miRNAs, which leads to resistance to apoptosis (28). Proteins associated with apoptotic pathways in cancer, including BAX in cervical cancer, caspase 3 in glioblastoma and squamous cell carcinoma, and caspase 9 in glioblastoma, have been studied previously (29,30). In the present study, the association between miR-21 and the PI3K/Akt pathway was investigated in order to elucidate the apoptotic mechanism.

The PI3Ks and Akts consist of multiple isoforms, and the PI3K/Akt signaling pathway is able to regulate cellular processes as diverse as cell growth, survival, proliferation and migration (31,32). Due to the function of signal transmission, PI3K/Akt has been associated with signaling pathways in various types of cancer, including lung cancer (33,34). Expression of p-Akt, Akt, Bcl-2, BAX, procaspase-3, active caspase-3 and actin proteins were measured by western blotting in the present study, in order to observe the underlying mechanism of the regulatory function of miR-21. The western blotting results demonstrated that the inhibition of apoptosis mediated by miR-21 in A549 cells is associated with activation of the PI3K/Akt signaling pathway. The expression of Akt was inhibited and the apoptotic rate was measured. The results of the present study demonstrated that the inhibition of Akt decreased the apoptotic rate of lung cancer cells.

The results of the present study demonstrated that the overexpression of miR-21 increased cell viability in lung cancer cells. In addition, the present study demonstrated the underlying mechanism of apoptosis in lung cancer. The results of the present study suggest that miR-21 may be involved in the progression of lung cancer and may be a novel therapeutic target for the disease, and the present results may provide a novel therapeutic approach to target lung cancer metastasis via the PI3K/Akt signaling pathway. The present study provides a foundation for further studies into the role of miR-21 in lung cancer. Specific aspects of the underlying mechanism require further research.

Acknowledgements

The present study was supported by the Demonstration Project of Ningbo Science and Technology Service Industry (grant no. 2014F10026).

References