MicroRNA-107 enhances gefitinib-induced growth inhibition in lung cancer cells

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MicroRNA-107 enhances gefitinib-induced growth inhibition through suppression of cyclin E1 and cyclin-dependent kinase 6 in non-small cell lung cancer cells

Running title

MiR-107 improves gefitinib efficacy in NSCLC

Highlights

  1. MiR-107 combined gefitinib significantly inhibited cell viability.
  2. MiR-107 combined gefitinib significantly arrested cell cycle at G1 phase.
  3. MiR-107 combined gefitinib significantly induced cell apoptosis.
  4. CCNE1 and CDK6 were markedly reduced in miR-107 transfected cells.

Abstract

Objective: we aimed to evaluate the effect and mechanism of microRNA-107 (miR-107) combined with gefitinib on cell viability, cycle and apoptosis in A549 cells.

Methods: Cells were divided into four groups: control group, gefitinib group, 30 nmol/L, and 60 nmol/L miR-107 combined gefitinib group. The gefitinib concentrations that caused 50% inhibition (IC50) were calculated. A tetrazolium-based colorimetric(MTT) assay and flow cytometry were used to investigate cell viability, cycle and apoptosis. Protein expressions were identified by quantitative real-time polymerase chain reaction and western blot.

Results: IC50 of gefitinib was significantly decreased from 17.31 ± 0.79 μmol/L to 12.18 ± 0.81 μmol/L (30 nmol/L, P < 0.05) and 9.11 ± 0.86 μmol/L (60 nmol/L, P < 0.01). Cell cycle was significantly arrested at G1 phase both in 30 nmol/L miR-107 group (49.62 ± 4.01, P < 0.05) and 60 nmol/L miR-107 group (70.18 ± 5.27, P < 0.05) compared to gefitinib (40.88 ± 3.21). Moreover, the apoptosis was significantly increased in 30 nmol/L miR-107 group (30.12 ± 6.34, P < 0.05) and 60 nmol/L miR-107 group (42.68 ± 7.99, P < 0.05) compared with gefitinib (12.11 ± 3.82). In addition, CCNE1 and CDK6 expressions were significantly reduced to 71.7% (30 nmol/L, P < 0.05) and 73.6% (60 nmol/L, P < 0.05), respectively.

Conclusions: MiR-107 may be a promising candidate to improve the efficacy of gefitinib in NSCLC cells, and the remarkable inhibition of proliferation may be related to suppression of CCNE1 and CDK6.

Keywords: Non-small cell lung cancer; miR-107; gefitinib; combination

Introduction

Non-small cell lung cancer (NSCLC) is one of the leading causes of death all over the world (1). It mainly consists of 3 subtypes: squamous cell carcinoma, adenocarcinoma, and large cell carcinoma. Currently, most NSCLC patients are diagnosed with advanced cancer (2). The treatment is often varied depending on the stage of cancer, the health condition of patients, and the response to chemotherapy. For patients with early/non-metastatic NSCLC, surgery combined with chemotherapy is the best choice, whereas for patients with advanced/metastatic NSCLC, several chemotherapies such as gefitinib are used (3).

Gefitinib, an orally administered tyrosine kinase inhibitor, targets to ATP cleft in the tyrosine kinase epidermal growth factor receptor (EGFR), which is over-expressed in many cancers (4). It inhibits the growth of some cancer cells and leads to regressions in tumor xenografts with the minimal adverse effects (5). Compared with docetaxel, gefitinib is suggested as the efficient treatment for pretreated patients with advanced NSCLC (6). However, chemotherapy is often considered insensitive to patients with NSCLC, and tumor responses caused by gefitinib are detected in only a small percent (< 20%) of patients with advanced NSCLC (7,8). Moreover, patients who are initially respond to gefitinib still develop progressive disease, finally (9). Thus, improving efficiency of gefitinib is still the major goal of researchers.

It has been reported that microRNAs (miRNAs) are associated with cellular differentiation, proliferation, and death. MiRNAs are frequently dysregulated in human cancers and act as oncogenes or tumor suppressor genes (10). Raponi et al. has described differentially expressed miRNAs in lung squamous cell carcinoma compared to normal lung tissues. High expression of miR-155 and low expression of let-7, miR-126, and miR-449 are suggested to predict poor prognosis of lung adenocarcinoma (11,12). MiRNA-126 is identified as a tumor suppressor gene, and its overexpression would reduce cell proliferation and inhibit tumor growth of the nude mouse xenograft model (13). MiR-449 has been demonstrated to induce G1 arrest, cell apoptosis, and senescence and the biological target genes have been partially identified (12). Besides, Takahashi and colleagues identified another two cell cycle regulating miRNAs: miR-107 and miR-185. Among them, miR-107 expression was lower in lung cancer cell lines compared with normal lung tissues, and it was suggested to regulate the downexpression of a large number of genes involving in cell cycle (14). Therefore, in our study, we investigated the effects of miR-107 overexpression combined with gefitinib on cell viability, cell cycle, and cell apoptosis. In addition, the downstream target genes regulated by miR-107 was also identified using quantitative real-time polymerase chain reaction (qRT-PCR) assay and western blot.

Materials and methods

Cell culture and reagent

Human lung adenocarcinoma A549 cells were stored in our laboratory and was cultured in Roswell Park Memorial Institute (RPMI-1640) medium (Invitrogen, Carlsbad, USA) supplemented with 10% fetal bovine serum (Hyclone, Logan, USA) in a humidified environment containing 5% CO2. MiR-107 sequence was downloaded from MirBase database (www.mirbase.com). Gefitinib (Iressa, AstraZeneca, UK) was dissolved in dimethyl sulfoxide (DMSO) to a concentration of 10 mmol /L and stored at -20 ˚С.

Cell transfection

Logarithmic A549 cells were digested by0.25% trypsin. A total of 500 µl cells were seeded in 24-well plates at 5×105 cells per well and incubated in RPIM-1640 medium for 24 h. Until the cell growth area was 60% to 70%, the plates were washed twice by RPIM-1640 medium without serum. Then, 30 nmol/L or 60 nmol/L fluorescein (FAM) -labeled small nuclear RNA U6 (RNU6B), miR-107, and negative control RNAs (synthesized by Genephama, Shanghai, China) were transiently transfected with A549 cells according to the protocol. For the detection of transfection efficiency, FAM-labeled control RNA transfected cells were incubated for 6 h and the medium was changed, and then cells were observed using fluorescent microscope (Nicon, Tokyo, Japan). The transfection efficiency was termed as the percentage ratio between the number of fluorescent cells and the number of normal cells. Each experiment was performed 3 times and the average was calculated. Therefore, cells were divided into four groups: control group (A549 cells), gefitinib group, 30 nmol/L miR-107 combined gefitinib group, and 60 nmol/L miR-107 combined gefitinib group.

qRT-PCR assay for miR-107

MiR-107 (60 nmol/L) transfected A549 cells were incubated for 48 h. Total RNAs were extracted using Trizol (Invitrogen, Carlsbad, USA) and then reversed into cDNA with One Step PrimeScript® miRNA cDNA Synthesis Kit (TaKaRa, Otsu, Japan). cDNA was quantified using SYBR PrimeScript RT-PCR Kit (TaKaRa, Otsu, Japan) basing on the manuscript’s instruction. RNU6B was used as an internal control. Total of 20 µl reactions was incubated for 30 min at 16 ˚С, 15 min at 37 ˚С, and 15 seconds at 85 ˚С to inactive reverse transcriptase. The primers used to synthesize cyclin E1 (CCNE1), cyclin-dependent kinase 6 (CDK6), and β-actin were listed in Table 1. The comparative cycle threshold (ΔΔCt) method (15) was used to determine the relative expression level of miR-107 compared with RNU6B or the relative level of CCNE1 and CDK6 compared with β-actin, respectively.

Cell viability analysis by MTT assay

The 3- (4, 5-Dimethylthiazol-2-yl) -2, 5-Diphenyltetrazolium bromide (MTT) assay was used to analyze the apoptosis rate of cells. Total of 100 µl logarithmic A549 cells were seeded in 96-well plates at 0.5-1×104 cells per well and incubated for 24 h. Then, 30 nmol/L or 60 nmol/L miR-107 and negative control RNA were transfected with A549 cells and kept overnight for attachment (24 h). Each group was independently repeated for 15 times. Subsequently, transfected cells were incubated with fresh medium containing different concentrations of gefitinib (0, 3, 6, 12, and 24 μmol/L) for 48 h. Total of 20 µl MTT (5 mg/ml, Sigma, Dorset, UK) was added and incubated for 4 h (37˚С, 5% CO2). After removal of the supernant, 150 µl DMSO was added and incubated for 10 min with shaking under 37 ˚С. The absorbance was detected at a wavelength of 490 nm using a colorimetric plate reader (Bio-Rad Laboratories, Hercules, USA). The inhibition rate was termed as the percentage value relative to the untreated control. Dose-dependent curves were plotted, and gefitinib concentrations that caused 50% inhibition (IC50) were calculated. Each experiment was performed three times independently.

Cell cycle and apoptosis analysis byflow cytometry and annexin V-FITC/PI

Flow cytometry was used to analyze cell cycle. MiR-107 or negative control RNA transfected cells were seeded in a 6-well plate at 0.5-1×104 cells per well and allowed to attach overnight. Fresh medium (without serum) was added in order to synchronize cells for 24 h, followed by treatment with gefitinib at a final concentration of 6 μmol/L and incubated for 12 h or 24 h, respectively. Then, cells were collected, washed by phosphate-buffered saline (PBS) for 2 times, and fixed in 75% ethanol. Supernant was discarded and cells were washed with PBS. Finally, 50 µl 0.1% Triton X-100 was added to the wells and incubated for 30 min in dark, and then cell cycle analysis was analyzed by flow cytometry (Becton-Dickinson, NJ, USA). Non-treated cells were used as negative control.

The annevin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) staining kit (BD Pharmingen, San Diego, USA) was used to assess the apoptotic death rate of cells incubated for 48 h. Cells were collected, washed by PBS, and resuspended in 500 µl binding buffer to a final concentration of 5×105 cells/mL. Then, annevin V-FITC labeling solution (5 µl) and PI solution (10 µl) were added and incubated for 15 min at 18~28 ˚С. Each experiment was repeated 3 times. Analysis was performed by flow cytometry (Becton-Dickinson, NJ, USA). Apoptotic cells were defined as annexin- FITC positive.

Western blot

Transfected cells treated with gefitinib (6 μmol/L) were harvest and lysed in RIPA buffer (25mM tris-HCl (pH 8.0), 1% Nonidet-P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), and 125mM NaCl) containing phenylmethylsulfonyl fluoride (PMSF, 1: 100) for 30 min at 4 ˚С. Total protein concentration was quantified using BCA protein assay (Pierce, Rockford, USA). Proteins were separated on a SDS-polyacrylamide gel electrophoresis and transformed to polyvinylidence fluoride (PVDF) membranes. Samples were incubated with anti-CCNE1 (1: 2000) or anti-CDK6 (1: 2000) overnight and then with horseradish peroxidase-conjugated second antibodies (1: 5000, Santa Cruz Biotechnology, Santa Cruz, USA). The results were detected through an enhanced chemiluminescence detection kit (Amersham Pharmacia Biotech, Buckinghamshire, UK). β-actin was used as a internal control.

Statistical analysis

Data were presented as mean±standard deviation (SD). Statistical differences were determined by student’s t test. P<0.05 was considered to be significantly different.

Results

Overexpression of miR-107 enhanced the cytotoxicity of gefitinib to A549 cells

The transfection efficiency of 30 nmol/L or 60 nmol/L RNU6B to cells was 79.1% and 88.6%, respectively. The expression analysis of miR-107 by qRT-PCR was shown in Fig. 1A. When transfected with 30 nmol/L or 60 nmol/L miR-107, the expression of miR-107 was significantly increased 5- or 8-fold compared with A549 cells.

The result of MTT assay was shown in Fig. 1B. The viability was decreasing with the rising concentration of gefitinib. There was a significant inhibition effect of miR-107 combined with gefitinib on cell viability compared to gefitinib alone. As shown in Table 2, the IC50 of gefitinib was 17.31 ± 0.79 μmol/L before transfection, whereas it was significantly decreased to 12.18 ± 0.81 μmol/L (P < 0.05) after transfected with 30 nmol/L miR-107, and significantly decreased to 9.11 ± 0.86 μmol/L after transfected with 60 nmol/L miR-107 (P < 0.01), as well.

MiR-107 combined with gefitinib treatment induced cell cycle arrest

Compared with gefitinib group, the percentages of cells in G0/G1 phase were significantly increased (P < 0.05, Table 3) either in 30 nmol/L miR-107 group combined gefitinib (49.62 ± 4.01 vs. 40.88 ± 3.21, P < 0.05) or in 60 nmol/L miR-107 combined gefitinib group (70.18 ± 5.27 vs. 40.88 ± 3.21, P < 0.05). Consistently, the percentages of S phase cells were decreased, and they were 31.96 ± 3.65 (gefitinib), 28.54 ± 3.28 (30 nmol/L miR-107), and 12.82 ± 2.59 (60 nmol/L miR-107), respectively.

MiR-107 combined with gefitinib treatment increased cell apoptosis

The apoptotic effects of gefitinib on A549 cells were shown in Fig. 2. Compared to control cells, the apoptotic death rate of gefitinib was (12.11 ± 3.82) %. After transfected with 30 nmol/L or 60 nmol/L miR-107, the apoptotic death rate was significantly increased to (30.12 ± 6.34) % (P < 0.05) and (42.68 ± 7.99) % (P < 0.05), respectively.

MiR-107 overexpression depressed the expressions of CCNE1 and CDK6

The expression analyses of CCNE1 and CDK6 were shown in Fig. 3. In miR-107 combined with gefitinib group, the mRNA expressions of CCNE1 and CDK6 were significantly reduced to 71.7% and 73.6% compared with gefitinib group (P < 0.05, Fig. 3A). Besides, the western blot demonstrated the same tendency (Fig. 3B). CCNE1 and CDK6 protein expressions were significantly decreased in miR-107 combined gefitinib group compared to control group.

Discussion

In the current study, miR-107 combined with gefitinib treatment significantly decreased cell viability, induced cell cycle arrest, and increased cell apoptosis in NSCLC cells. Furthermore, we demonstrated that the expressions of CCNE1 and CDK6 involving in cell cycle were significantly reduced in miR-107 transfected cells.

The dysregulation of EGFR promotes tumor growth by increasing cell proliferation, migration, invasive ability, and by inhibiting cell apoptosis (16). EGFR is over-expressed in NSCLC, and has been identified as a promising target for anticancer therapy (17). Gefitinib is recently used to treat advanced or metastatic NSCLC patients with activating mutations in the tyrosine kinase domain of EGFR(18). However, Han et al. (9) has demonstrated that Marsdenia tenacissima extract could improve the efficacy of gefitinib in NSCLC cells regardless of EGFR status. Besides, studies reported that gefitinib has no significant effect on cell cycle progression and cell apoptosis in A549 cells, even treated with 10 µmol/L gefitinib (19,20). Therefore, in the present study, we analyzed the effect of miR-107 overexpression on the efficacy of gefitinib to NSCLC cells viability and proliferation. Interestingly, the results suggested that overexpression of miR-107 improved cell cytotoxicity of gefitinib to A549 cells with IC50 at 12.18 ± 0.81 μmol/L (30 nmol/L) and 9.11 ± 0.86 μmol/L (60 nmol/L), respectively. Furthermore, flow cytometric data revealed that miR-107 overexpression combined with gefitinib significantly induced G1/S arrest and cell apoptosis in A549 cells. This result is consistently with the effect of miR-107 overexpression alone (14). Taken together, these findings suggest that miR-107 overexpression in combination with gefitinib could inhibit the growth of A549 cells through enhancing cell cycle arrest and cell apoptosis.

High expression of CCNE1 protein is often observed in many cancers, including ovarian cancer (21), breast cancer (22), gastric cancer (23), as well as lung cancer (24). High expression of CCNE1 is often associated with poor prognosis in cancer patients. It has been shown that the activity of CCNE1 contributes to cell cycle G1/S transition. It is accumulated at G1/S phase and degraded when cells progress through S phase (25). Silencing of CCNE1 by siRNA leads to cell cycle arrest in liver cancer cell lines (26). Besides, CDK6 is a member of serine-threonine kinase family and also has a regulatory role in G1/S transition via phosphorylation of retinoblastoma protein (27,28). For example, knockout of CDK6 with siRNA results in prolonged S phase in human embryonic stem cells (29). Moreover, in hepatocellular carcinoma, down-regulation of miR15/16/195 family member results in downexpression of cell cycle related proteins, such as CDK6 and CCNE1, and caused an accumulation of G1 phase cells (30). Previous study reported that overexpression of miR-107 suppressed the expression of CDK6 in human pancreatic cancer cell lines (31). In addition, in NSCLC cells, Takahashi and colleagues have suggested that down-regulated genes by miR-107 are primarily associated with cell cycle using Go Ontology analysis, and these down-regulated genes involve CCNE1 and CDK6 (14). In our study, CCNE1 and CDK6 were consistently down-expressed in vivo in A549 cells treated by miR-107 and gefitinib. Therefore, we suggest that the enhanced cytotoxicity of gefitinib by miR-107 is achieved by decreasing the expressions of CCNE1 and CDK6.

In conclusion, we have found that overexpression of miR-107 enhances cell cytotoxicity of gefitinib to A549 cells through decreasing cell viability, inducing cell cycle arrest, and increasing cell apoptosis. Meanwhile, the underlying mechanism of this remarkable inhibition of proliferation may be related to suppression of CCNE1 and CDK6. Thus, miR-107 may be a promising candidate to improve the efficacy of gefitinib in NSCLC cells. However, the effect of miR-107 combined gefitinib to other NSCLC cell lines like HCC827 or H292, and the association between miR-107 combined gefitinib and EGFR status still needs further research.

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