The A549 cell line was purchased from the American Type Culture Collection (Manassas, VA, USA). The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with Glutamax (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Hyclone; GE Healthcare Life Sciences; Logan, UT, USA), 100 U/ml penicillin and 0.1 mg/ml streptomycin. Cells were cultured until they reached 80% confluence, following which the medium was replaced with serum-free DMEM and the cells were incubated for a further 15 h at 37°C in a humidified atmosphere containing 5% CO₂. The cells were then stimulated with 10 µg/ml of Escherichia coli-derived LPS (O26:B6; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) for 6 h in a humidified atmosphere containing 5% CO2 at 37°C (10).

The RhoA-specific siRNA (si-RhoA) (sense: CCUUAUAGUUACUGUGUAATT; antisense: UUA CAC AGU AAC UAU AAA GGT A) and the negative control (si-NC) (sense: UUA UCG CCA AAU UCU UUU AUC GGA CAG AG; antisense: UUG AUA AAA GAA UUU GGC GAU GGA CAG AG), which had no silencing effect, were synthesized by Shanghai GenePharma Co., Ltd. (Shanghai, China). Cells were seeded into 100 mm dishes at a concentration of 10⁶ cells/dish and incubated at 37°C in a humidified atmosphere containing 5% CO₂ for 24 h. Then, 25 mg/well of a RhoA specific or a negative control siRNA was diluted into Lipofectamine 3000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and added to cells. After approximately 2 weeks, stable RhoA transfections were generated under G418 (Gibco; Thermo Fisher Scientific, Inc.) selection as described (17).

Cell proliferation was measured using the MTT colorimetric assay. In brief, non-infected cells and cells transfected with si-RhoA or si-NC were seeded in 96-well plates at a concentration of 10⁴ cells/well in a humidified atmosphere containing 5% CO₂. Cells were incubated in normal condition for 1, 2, 3 and 4 days prior to being treated with LPS for 6 h. Treated cells were washed twice with PBS and added with MTT (5 mg/ml) (Sigma-Aldrich; Merck KGaA) in the dark and incubated at 37°C for another 3 h. Then, 100 µl DMSO was added to dissolve the formazan crystals. The optical density of each well was measured at a wavelength of 570 nm on a microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

ROS activity was measured by flow cytometry using 2′,7′-dichlorofluorescein diacetate (DCFH-DA; Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The cells were seeded into a 6-well plate, at a concentration of 10⁵ cells/ml for 24 h and were then exposed to LPS for 6 h. The treated cells were washed twice with PBS and were incubated in serum-free culture medium containing 10 µM DCFH-DA for 20 min at 37°C in the dark. The cells were then washed with PBS, and collected using the trypsin digestion method. All samples were centrifuged at 800 × g for 5 min at 4°C and the supernatants were removed. The cells were resuspended in 500 µl PBS and fluorescent intensities were measured using a flow cytometer.

An apoptosis analysis was performed by flow cytometry using the Annexin V-fluorescein isothiocyanate/propidium iodide (PI) apoptosis detection kit (Beijing Biosea Biotechnology Co., Ltd., Beijing, China). The treated A549 cells were washed twice with cold PBS and resuspended in 1X binding buffer. The adherent and floating cells were combined and were treated according to the manufacturer's protocol. Cells were then measured using a flow cytometer (Beckman Coulter, Inc., Brea, CA, USA) to differentiate apoptotic cells (Annexin-V⁺ and PI⁻) from necrotic cells (Annexin-V⁺ and PI⁺).

Total RNA was isolated from transfected cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and was treated with DNase I (Promega Corporation, Madison, WI, USA). RT was performed using the Multiscribe RT kit (Applied Biosystems; Thermo Fisher Scientific, Inc.) and random hexamers or oligo(dT). The RT conditions were as follows: 10 min at 25°C, 30 min at 48°C and a final step at 95°C for 5 min (18). The expression of RhoA, Wnt3a, β-catenin, Wnt5a, Bcl-2, pro-caspase-3 and cleaved caspase-3 were conducted by qRT-PCR using the SYBR Green Master Mix (Takara, Dalian, China) according to manufacturer's protocols. The PCR primers were obtained from Invitrogen (Thermo Fisher Scientific, Inc.), and the reaction conditions included the following steps: 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min. The sequences of the primers used for qPCR were as follows: RhoA (forward: 5′-CTCATAGTCTTCAGCAAGGACCAGTT-3′, reverse: 5′-ATCATTCCGAAGATCCTTCTTATT-3′), Wnt3a (forward: 5′-GCAGTTGCGAAGTGAAGACC-3′, reverse: 5′-TGTGGGCACCTTGAAGTATGT-3′), β-catenin (forward: 5′-GATTAACTATCAGGATGACGCG-3′, reverse: 5′-TCCATCCCTTCCTGCTTAGTC-3′), Bcl-2 (forward: 5′-GAGTGGGATGCGGGAGATGTG-3′, reverse: 5′-AGCGGCGGGAGAAGTCGTC-3′), Wnt5a (forward: 5′-CGAAGACAGGCATCAAAGAA-3′, reverse: 5′-GCAAAGCGGTAGCCATAGTC-3′), Pro-caspase-3 (forward: 5′-CTGGACTGTGGCATTGAG-3′, reverse: 5′-GCTTGTCGGCATACTGTT-3′), Cleaved caspase-3 (forward: 5′-TGGTTCATCCAGTCGCTTTG-3′, reverse: 5′-ATTCTGTTGCCACCTTTCGG-3′) and GAPDH (forward 5′-GCACCGTCAAGGCTGAGAAC-3′, reverse: 5′-TGGTGAAGACGCCAGTGGA-3′). The mRNA of GAPDH level was used as an internal control and relative expression changes were calculated using the 2^−ΔΔCq method (19).

Protein was extracted using radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China) supplemented with protease inhibitors (Roche Diagnostics, Shanghai, China), and was quantified using the Bicinchoninic Acid Protein Assay kit (Pierce; Thermo Fisher Scientific, Inc.). Electrophoresis was conducted using a Bio-Rad Bis-Tris Gel system (Bio-Rad Laboratories, Inc., Hercules, CA, USA) according to the manufacturer's protocol. Denatured protein (30 µg) was loaded onto 15% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE), electrophoresed, and then transferred onto polyvinylidene fluoride (PVDF) membranes (EMD Millipore, Billerica, MA, USA). Membranes were blocked with 5% BSA for 2 h at room temperature. RhoA antibody (sc-179) was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA) and GAPDH antibody (G8795) was purchased from Sigma-Aldrich; Merck KGaA. Other primary antibodies were: anti-Wnt3a (2721), anti-β-catenin (8480), anti-Cleaved caspase 3 (9662), anti-Wnt5a (2530) and anti-Bcl-2 (4223) purchased from Cell Signaling Technology (Danvers, MA, USA). The primary antibodies were prepared in 5% BSA at a dilution of 1:1,000. The polyvinylidene fluoride membranes were incubated with the primary antibodies at 4°C overnight, followed by washing with TBST (0.1% Tween) and incubation with a goat anti-rabbit IgG secondary antibody HRP conjugated (sc-2004; 1:5,000; Santa Cruz Biotechnology, Inc.) or a goat anti-mouse IgG secondary antibody HRP conjugated (sc-2005; 1:5,000; Santa Cruz Biotechnology, Inc.) in 5% BSA for 1 h at room temperature. Following rinsing, the blots were transferred to a Bio-Rad ChemiDoc™ XRS system (Bio-Rad Laboratories, Inc.) and 200 µl Immobilon Western Chemiluminescent HRP Substrate (EMD Millipore) was added to cover the membrane surface. The signals were captured and analyzed using Image Lab™ software version 4.0 (Bio-Rad Laboratories, Inc.).

The results of multiple experiments are presented as the mean ± standard deviation of three independent determinations. Statistical analyses were performed using SPSS 19.0 statistical software (IBM Corp., Armonk, NY, USA). P-values were calculated using a one-way analysis of variance followed by Tukey's multiple comparison tests. P<0.05 was considered to indicate a statistically significant difference.

A549 cells were treated with LPS to stimulate inflammatory injury. As shown in Fig. 1A and B, LPS increased the mRNA and protein expression levels of RhoA.

The expression of RhoA in A549 cells was knocked-down by siRNA. The results of RT-qPCR and western blotting confirmed that transfection with si-RhoA significantly decreased RhoA expression (P<0.05; Fig. 2A and B).

The MTT assay was used to determine the effects of RhoA on cell viability. The experimental group was treated with LPS and transfected with si-RhoA. LPS decreased cell viability; however, A549 cells treated with LPS and si-RhoA exhibited increased cell viability compared with the LPS + siNC group (Fig. 3).

The present study also aimed to determine the role of RhoA in ROS activity. A549 cells were treated with LPS and were then transfected with si-RhoA. Compared with in the control group, ROS activity was significantly increased (P<0.01) in cells exposed to LPS. However, ROS activity was decreased following si-RhoA transfection. These findings suggested that RhoA knockdown may prevent LPS-induced increases in ROS activity (Fig. 4).

The effects of RhoA knockdown on cell apoptosis were determined by flow cytometry. The results demonstrated that LPS significantly increased cell apoptosis (P<0.01), whereas RhoA knockdown protected cells from LPS-induced apoptosis (Fig. 5). Therefore, it may be hypothesized that inhibition of RhoA protects A549 cells from cell injury by inhibiting apoptosis.

As presented in Fig. 6, the mRNA and protein expression levels of Wnt3a, β-catenin, Wnt5a, Bcl-2, pro-caspase-3 and cleaved caspase-3 were detected. The results demonstrated that RhoA knockdown may inhibit the activation of the downstream Wnt/β-catenin signaling pathway and inhibit the expression of apoptotic factors. Therefore, knockdown of RhoA may protect cells from LPS-induced damage via the Wnt/β-catenin signaling pathway.