Everolimus was purchased from Novartis Pharma AG (Basel, Switzerland), and anti-PTEN (cat. no. 9188; 1:1,000) and anti-β-actin (cat. no. 3700; 1:5,000) antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Prior to addition to cell cultures, everolimus was prepared in dimethyl sulfoxide (DMSO) at a stock concentration of 10 mM.

A549 cells were purchased from the American Type Culture Collection (Manassas, VA, USA) and were cultured in RPMI-1640 medium (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.), 50 U/ml penicillin and 50 µg/ml streptomycin. All culture supplements were by Invitrogen (Thermo Fisher Scientific, Inc.) and cells were maintained at 37°C with 5% CO₂.

Cells were treated at 37°C for 72 h with everolimus at 50 or 100 nM or a vehicle(0.01% DMSO) as a negative control.

A549 cells were plated at a density of 1×10⁵ cells/well, and allowed to adhere overnight in a 6-well dish, and the cells were transfected with PTEN small interfering RNA (siRNA), miR-4328 mimics or non-specific negative control, which were synthesized by GenePharma (GenePharma, Shanghai, China), using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocols. The sequences were as follows: PTEN siRNA, 5′-GGCUAAGUGAAGAUGACAATT-3′; miR-4328 mimics, 5′-CCAGUUUUCCCAGGAUU-3′; and non-specific negative control, 5′-UUCUCCGAACGUGUCACGUTT-3′. At 24 h following transfection, everolimus was used to treat the transfected A549 cells, as aforementioned.

Cellular proliferation ability of A549 cells was measured by Cell Counting Kit-8 (CCK-8, Dojindo Laboratories, Kumamoto, Japan), according to the manufacturer's protocol. At 24 h following transfection, cells were seeded into 96-well plates at a density of 8×10³ cells/well with 100 µl cell culture medium (with 10% FBS), and at the indicated time points (days 1, 2, 3, 4 and 5), 10 µl CCK-8 was added to each well. The absorbance at 450 nm was detected by a plate reader following incubation for 1 h at 37°C. The experiments were repeated three times.

The migration abilities of A549 cells were analyzed with a Transwell assay, as described previously (23). Briefly, the cells were digested and seeded into the upper chamber (Transwell inserts) at a density of 3×10⁵ cells/ml with cell culture medium containing 10% FBS. Cell culture medium supplemented with 20% FBS was added to the lower chamber, and incubated at 37°C for 36 h. Following this incubation, the membrane was stained with 0.1% crystal violet for 30 min at room temperature. These stained cells were counted using a light microscope following washing with PBS at ×20 magnification. The experiments were performed three times.

pmiR-PTEN 3′untranslated region (UTR) wild-type or pmiR-PTEN 3′UTR mutant were transfected into A549 cells with pRL-TK vectors (Promega Corporation, Madison, WI, USA) with Lipofectamine 2000 for 24 h. Subsequently, 100 nM everolimus was added to treat the transfected A549 cells for 48 h at 37°C. Using a Dual Luciferase Reporter assay system (Promega Corporation), the luciferase activity was measured with a microplate luminometer (Infinite F200; Tecan, Männedorf, Switzerland) and the relative luciferase activity was normalized to firefly luciferase activity. The experiments were repeated three times.

Total RNA was isolated from the A549 cells using the RNeasy Mini kit (Qiagen GmbH, Hilden, Germany), according to the manufacturer's protocol, and used for RT-qPCR. To detect the relative expression levels of PTEN, RT-qPCR was performed. The total PCR volume was 20 µl and consisted of 10 µl 2X Power SYBR^® Green PCR Master mix (Applied Biosystems; Thermo Fisher Scientific, Inc.), 2 µl cDNA (5 ng/µl) and 1 µl primer mix (10 µM each). Using the LightCycler 480 II (Roche Applied Science, Penzberg, Germany) to perform PCR amplification and the procedure for PCR amplification was as follows: Denaturation at 95°C for 10 min, 40 cycles of 95°C for 15 sec, and then 60°C for 1 min. The comparative Cq method was used to calculate the relative gene expression level (24), and the expression levels of detected gene were normalized to an endogenous reference GAPDH. Additionally, those relative to the calibrator were provided by the formula 2^−ΔΔCq (25). With the use of the Hairpin-it™ miR-4328 qRT-PCR Primer Set (Shanghai GenePharma, Co., Ltd., Shanghai, China), the relative quantity of miR-4328 was measured. The primers were as follows: PTEN forward, 5′-TGGATTCGACTTAGACTTGACCT-3′ and reverse, 5′-GGTGGGTTATGGTCTTCAAAAGG-3′; GAPDH forward, 5′-GGAGCGAGATCCCTCCAAAAT-3′ and reverse 5′-GGCTGTTGTCATACTTCTCATGG-3′. The experiments were performed three times.

Cultured A549 cells were harvested and lysed with radioimmunoprecipitation assay buffer (Beijing Solarbio Science and Technology Co., Ltd., Beijing, China) was subsequently added, and cells were incubated on ice for protein extraction for 30 min. The protein concentration was measured by the bicinchoninic acid method (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). Equal amounts of proteins (10 µg per sample) were separated by SDS-PAGE (12% gels) and transferred onto polyvinylidene fluoride membranes, which were blocked in 10% skimmed milk in PBS containing 0.1% Tween-20 for 2 h at room temperature. The membranes were probed overnight at 4°C with the aforementioned anti-PTEN and anti-β-actin primary monoclonal antibodies and in horseradish peroxidase (HRP)-conjugated secondary antibodies (rabbit anti-mouse and mouse anti-rabbit IgG; cat. nos. 58802 and 93702, respectively; 1:2,000; Cell Signaling Technology) for 1 h at 25°C. Using an enhanced chemiluminescence reagent (Sigma-Aldrich; Merck KGaA) to detect the target, β-actin was used as the normalization control. ImageJ software version 1.41 (National Institutes of Health, Bethesda, MD, USA) was used for the densitometry analysis. The experiments were performed three times.

Data was analyzed by GraphPad Prism version 5.01 (GraphPad Software, Inc., La Jolla, CA, USA) and presented as mean ± standard deviation. For multiple comparison analysis, one-way analysis of variance followed by Tukey's multiple comparison post-test was performed. P<0.05 was considered to indicate a statistically significant difference.

The present study treated A549 cells using everolimus at concentrations of 50 and 100 nM, and the PTEN expression was observed to be significantly enhanced (P<0.01), and the expression of the 100 nM group was increased, compared with the 50 nM group (Fig. 1A and B). The effects of everolimus on the cell proliferation and migration of lung cancer cells were also detected, and everolimus was demonstrated to significantly inhibit the proliferation and migration of A549 cells at 50 and 100 nM (P<0.05; Fig. 1C-E). The everolimus treatment also significantly upregulated the mRNA levels of PTEN at 50 and 100 nM (P<0.001; Fig. 1F).

In order to investigate the roles of PTEN in the therapeutic process of everolimus on the A549 cells, transfection experiments were performed to knockdown the expression of PTEN and examine the effects of everolimus treatment. PTEN siRNA significantly reduced the expression of PTEN following everolimus treatment (P<0.001; Fig. 2A and B). Notably, it was identified that the knockdown of PTEN significantly enhanced the proliferation of A549 cells in the everolimus-treated samples (P<0.05; Fig. 2C). The present study also identified that silencing of PTEN reduces the effects of everolimus treatment on the migration of A549 cells (Fig. 2D and E).

The present study further demonstrated that everolimus significantly downregulated the expression of miR-4328, which has previously been predicted as the candidate upstream regulatory molecule for PTEN (19) (Fig. 3A). To confirm the association between miR-4328 and PTEN, a luciferase reporter assay was performed and treatment with everolimus significantly decreased the reporter activity in A549 cells following transfection with a wild-type vector (P<0.001; Fig. 3B). Additionally, when transfection with a mutant vector was performed, the reporter activity was not significantly altered between the everolimus treatment and negative control groups (Fig. 3B). The everolimus-treated A549 cells were additionally transfected with miR-4328 mimics, and the upregulation of PTEN induced by everolimus was inhibited at the mRNA and protein levels (Fig. 3C and D). In order to confirm the role miR-4328 in the treatment of everolimus, miR-4328 mimics were transfected into cells treated with everolimus, and the results demonstrated that overexpression of miR-4328 significantly reduced the effects of everolimus on the proliferation and migration of A549 cells (Fig. 3E-G).