In total, 100 patients (43 males and 57 females; median age, 63 years) with pathologically confirmed advanced NSCLC admitted to the First Hospital of Lanzhou University (Lanzhou, China) between March 2012 and May 2014 were enrolled in the present study. Additional demographic data, including age, gender, histological type of tumor and clinical stage, are shown in Table I. All patients were administered with gefitinib orally (250 mg/day) and the effect of gefitinib was evaluated according to the Response Evaluation Criteria in Solid Tumors (RECIST) guidelines. In brief, the diameter of the tumors were calculated and analyzed, and patients were assessed for complete response (CR), partial response (PR), stable disease (SD) or progressive disease (PD). The tumor tissue samples were collected and stored in liquid nitrogen for additional analysis. Plasma samples were collected prior to and subsequent to therapy. The experimental protocols were approved by the Ethics Committee of The First Hospital of Lanzhou University, and all patients provided informed consent.

The human lung adenocarcinoma A549 cell line was obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured at 37°C in a 5% CO₂ atmosphere. All cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA).

The microRNA of plasma specimens and tissue samples were extracted with the MiRcute miRNA Extraction-Separation kit [Tiangen Biotech (Beijing) Co., Ltd, Beijing, China], according to the manufacturer's protocol. Following measurement of the purity and concentration, reverse transcription of microRNA to cDNA was assessed using One Step PrimeScript miRNA cDNA Synthesis kit [Takara Biotechnology (Dalian) Co., Ltd., Dalian, China]. The reverse transcription system and conditions were also followed in accordance with the manufacturer's protocol; a no cDNA control and a no reverse transcription control were also used in the same reaction system. The obtained cDNA was amplified by RT-qPCR using SYBR^® Premix Ex Taq™ II [Takara Biotechnology (Dalian) Co., Ltd.]. The reaction system and conditions were in accordance with the manufacturer's protocol. The microRNA-200b upstream primer sequence was 5′-GCGGCTAATACTGCCTGGTAA-3′. For normalizing the data of plasma microRNA, microRNA-16 was used as reference (upstream primer, 5′-CGCGCTAGCAGCACGTAAAT-3′). For normalizing the data of tissue microRNA, U6 small nuclear RNA (snRNA) was used as a reference (upstream primer, 5′-CTCGCTTCGGCAGCACA-3′). All downstream primers were universal 3′ miRNA primers. The relative expression level of microRNA-200b was calculated by the 2^−ΔΔCq method (16,17), ΔCq=Cq_target-Cq_reference.

The microRNA-200b mimic was designed and synthesized by Invitrogen (Thermo Fisher Scientific, Inc.). Prior to transfection, A549 cells were routinely maintained and grew to 70% confluency. Subsequently, 100 nM microRNA-200b mimic and microRNA negative controls (NC) were transfected into cells by N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl-sulfate reagent (Roche Applied Science, Mannheim, Germany), according to the manufacturer's protocol. The transfection efficiency was assessed by RT-qPCR in a preliminary experiment using a similar protocol to the aforementioned protocol. For RT-qPCR analysis, the data in cells were normalized by U6 snRNA with the upstream primer sequence 5′-CTCGCTTCGGCAGCACA-3′.

Transfected and non-transfected A549 cells were plated at a density of 1.0×10⁵ cells/well into a 96-well plate and incubated for 24 h at 37°C in a 5% CO₂ atmosphere. Subsequent to being maintained for 24 h, all cells were treated with 0.1 µM gefitinib for 6, 12, 24 and 48 h. Subsequent to gefitinib treatment, 10 µl CCK-8 reagent (Dojindo Laboratories, Kumamoto, Japan) was immediately added to each well. After 4 h, the intensity of staining was monitored by SpectraMax i3 microtiter plate spectrophotometer (Molecular Devices, LLC, Sunnyvale, CA, USA) at 450 nm and the cell viability was estimated.

Transfected and non-transfected A549 cells were treated with 0.1 µM gefitinib for 24 h and harvested. For cell cycle analysis, cells were fixed in 70% ethanol for 12 h and then incubated with bromodeoxyuridine (BrdU) and 7-amino-actinomycin D (7-AAD), according to the detailed protocol provided by BD Pharmingen BrdU Flow Kits (BD Biosciences, Heidelberg, Germany). The cell cycle was analyzed using FACSCalibur flow cytometer system (BD Biosciences). For apoptosis analysis, double staining (Annexin V-fluorescein isothiocyanate and propidium iodide) was performed and cells were also analyzed using the FACSCalibur flow cytometry system. The flow cytometry data were analyzed by CellQuest Pro software (BD Biosciences).

Using 24-well Transwell chambers (Corning Incorporated, Corning, NY, USA) the migration of A549 cells was observed. According to the manufacturer's protocol, the lower chambers were filled with 500 µl DMEM supplemented with 10% FBS, and 1×10⁴ cells and 100 µl DMEM without FBS were placed into the upper chambers. As routinely, Cells were cultured for 24 h at 37°C in the presence of 5% CO₂ and then the cells that had invaded the lower surface of the microporous membrane of the Transwell chamber were fixed using methanol. Subsequent to being stained with crystal violet, the cells were quantified.

Transfected and non-transfected A549 cells were cultured for 24 h; the total RNA in cells was extracted by TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). RNA was reverse-transcribed to cDNA using PrimeScript^® RT reagent kit [Takara Biotechnology (Dalian) Co., Ltd.]. The reverse transcription system was in accordance with the protocols of the kit. The synthesized cDNA was amplified by SYBR^® Premix Ex Taq™ II kit [Takara Biotechnology (Dalian) Co., Ltd.]. Primers are shown in Table II, The reaction system and program was in accordance with the protocols of the kit. The relative expression of target IGF-1 mRNA was analyzed by the 2^−ΔΔCq method (ΔCq=Cq_target-Cq_GAPDH).

Cells were lysed in radioimmunoprecipitation assay buffer on ice for 30 min and total proteins were extracted. In total, 25 µg of protein was boiled for 5 min, separated using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane. Subsequent to being blocked with 5% nonfat dry milk in Tris-buffered saline and Tween 20, membranes were probed with anti-IGF-1R (dilution, 1:100; catalog no. ab39398), anti-AKT (dilution, 1:500; catalog no. ab8805), anti-ERK (dilution, 1:1,000; catalog no. ab196883), anti-phospho-AKT (dilution, 1:500; catalog no. ab8933), phospho-ERK (dilution, 1:100; catalog no. ab214362) and anti-β-actin (dilution, 1:10,000; ab8227) antibodies (Abcam, Cambridge, UK) overnight at 4°C. Subsequent to washing, membranes were incubated with Goat Anti-Rabbit IgG H&L (HRP) secondary antibody (dilution, 1:2,000; catalog no. ab97051) for 1 h. Incubated membranes were treated with ECL reagent (Applygen Technologies, Inc., Beijing, China). Please state the host and target animals, clonality, dilution and catalog number for the antibodies.

All data are reported as the mean ± standard deviation. The statistical significance of the differences was analyzed by one-way analysis of variance or least significant difference-t test using SPSS 15.0 software (SPSS, Inc., Chicago, IL, USA). For non-parametric statistics, Mann-Whitney U test was performed for comparison between the two groups. P<0.05 was considered to indicate a statistically significant difference.

According to the RECIST guidelines, 7 patients were classified as CR, 30 as PR, 28 as SD and 35 as PD. The present study defined CR+PR patients as gefitinib-sensitive patients (n=37) and SD+PD patients as gefitinib-insensitive patients (n=63). As shown in Table I, the age, gender, clinical stage and history of treatments showed no statistical difference between CR+PR patients and SD+PD patients. RT-qPCR results showed that the plasma microRNA-200b levels in SD+PD patients were lower compared with the CR+PR patients (P=0.0068), and the microRNA-200b levels in tissue samples of gefitinib-insensitive patients were also lower, compared with gefitinib-sensitive patients (P=0.0000075) (Figs. 1 and 2). It is suggested that detecting microRNA-200b level may reflect the sensitivity of NSCLC patients to gefitinib.

Subsequent to upregulating the microRNA-200b level of A549 cells through transfection with a microRNA-200b mimic, the present study found that the anti-proliferative effect of gefitinib on A549 cells increased. For the non-transfected A549 cells 24 h subsequent to treatment with 0.1 µM gefitinib, the cell inhibition rate was 20.8±3.2%, and for the A549 cells transfected with microRNA-200b mimic, the inhibition rate increased to 45.6±4.3%, showing a statistically significant difference (P=0.0071). However, transfection with microRNA NC did not affect the inhibiting effect of gefitinib on A549 cells (Fig. 3).

Following a time period of 24 h subsequent to treatment of A549 cells using 0.1 µM gefitinib, the A549 cell apoptosis rate without transfection treatment was 18.3±2.9%, while the A549 cell apoptosis rate with upregulation of microRNA-200b was 29.2±3.1%, which was significantly increased compared with non-transfected A549 cells (P=0.032), as shown in Fig. 4. Cell cycle analysis showed that, subsequent to treatment with 0.1 µM gefitinib, the G₀/G₁ percentage of untransfected A549 cells was 38.6±4.2% and the G₂/M percentage was 15.6±2.3%, suggesting that gefitinib could cause G₀/G₁ arrest of A549 cells. Following treatment of the A549 cells with upregulation of microRNA-200b with gefitinib, the G₀/G₁ percentage was 70.4±7.9% and the G₂/M rate was 8.2±0.9%, indicating that upregulation of microRNA-200b could enhance arrest of G₀/G₁ phase induced by gefitinib. Similarly, the effect of microRNA NC on the cell cycle was not observed (Fig. 5).

As shown in Fig. 6, the Transwell chamber assay revealed that, following upregulation of microRNA-200b, gefitinib exhibited a markedly increased migration inhibition on A549 cells, which was manifested by a significantly reduced number of cells invading the lower surface of the polycarbonate membranes. The microRNA NC did not affect the migration inhibition ability of gefitinib.

RT-qPCR analysis revealed that, following the upregulation of the microRNA-200b level of A549 cells, the IGF-1R mRNA level in the cells was significantly reduced (P=0.012), but the mRNA levels of AKT and ERK were not changed (Fig. 7). The western blot analysis results were similar to those of RT-qPCR. The IGF-1R protein level was reduced, and AKT and ERK protein levels were not changed, but the levels of phosphorylated AKT and ERK were reduced (Fig. 8). The microRNA NC showed no effect on IGF-1R expression and AKT or ERK phosphorylation in A549 cells.