Long non-coding RNA receptor activator of nuclear factor-κ B ligand promotes cisplatin resistance in non-small cell lung cancer cells.

Non-small cell lung cancer (NSCLC) is a common malignancy associated with poor clinical outcomes and high mortality rate. The association between NSCLC development and long non-coding RNA (lncRNA) expression remains to be elucidated. The current study investigated the role of a novel lncRNA, receptor activator of nuclear factor-κ B ligand (RANKL), in the resistance of NSCLC to chemotherapy. RANKL expression was assessed via reverse transcription-quantitative PCR, cell death rate was evaluated using flow cytometry and sensitivity of cisplatin (DDP)-resistant A549/DDP cells to chemotherapy was determined using the Cell Counting Kit-8 assay. Western blotting was performed to quantify p53 protein levels. Compared with matched A549 cells, A549/DDP cells exhibited significant upregulation of RANKL expression. Sensitivity of A549/DDP cells to DDP was restored following RANKL knockdown. A549 cells overexpressing RANKL exhibited notably impaired DDP sensitivity compared with controls. Conversely, downregulated RANKL expression triggered cell death and inhibited cell migration via p53 stimulation and phosphatidylinositol 3-kinase/protein kinase B pathway suppression. The current findings indicate that RANKL contributes to DDP resistance in NSCLC and may represent a novel therapeutic target in this malignancy. Copyright: © Zhu et al.

Introduction

Non-small cell lung cancer (NSCLC) is associated with poor clinical outcomes and a notably high mortality rate (1). Despite notable progress in diagnosis and treatment, NSCLC recurrence and mortality rates remain high with a 5-year survival rate of <15% worldwide (2). Platinum-based chemotherapy agents, particularly cisplatin (DDP), are utilized in adjuvant therapy following surgical treatment of NSCLC (3). However, the efficacy of DDP treatment may be impaired due to resistance (4). Therefore, a further understanding of DDP resistance in NSCLC is crucial for the development of effective approaches to reduce resistance and improve NSCLC treatment.

Long non-coding RNAs (lncRNAs) are RNA transcripts that lack functional coding (5). They are comprised of >200 nucleotides (6) and serve as essential mediators of gene expression at various concentrations via chromatin remodeling and transcriptional, post-transcriptional and -translational modifications (7). Previous studies have demonstrated that abnormal lncRNA expression serves an essential role in various cellular processes, such as epigenetic modulation, alternative splicing and genomic imprinting (8-10). Moreover, lncRNAs participate in various physiological and pathological aspects of cancer, including development, metastasis and invasion (11-14). Furthermore, lncRNAs are associated with treatment resistance in various cancers, including colon cancer, gastric cancer, chronic myeloid leukemia, NSCLC and ovarian cancer (15-19). Consequently, a further understanding of their role in the development of cancer and treatment resistance is warranted. Pre-experimental data indicated that the expression of novel lncRNA receptor activator of nuclear factor-κ B ligand (RANKL) increases in DDP resistance in NSCLC cells. However, the exact role of RANKL in NSCLC cells remains unclear.

Therefore, the aim of the current study was to investigate the expression of RANKL and its role in DDP resistance in NSCLC cells.

Materials and methods

Cell culture

NSCLC A549 and A549/DDP cells were purchased from the Chinese Academy of Sciences and cultured on RPMI-1640 supplemented with 10% FBS (both from Gibco; Thermo Fisher Scientific, Inc.) at 37˚C with 5% CO2.

Lentiviral vector construction and transfection

The lncRNA RANKL sequence (5'-CAGAAGATGGCACTCACTGCA-3') was generated by Genewiz, Inc. Recombination was achieved using temporary calcium phosphate-mediated transfection of 293T cells (Cell Bank of Type Culture Collection of Chinese Academy of Sciences). The lentiviral vector was subcloned using plasmids and cells were transfected with these plasmids using a Lentiviral Packaging mix (packaging vector: Envelope=1:10) (Shanghai GenePharma Co., Ltd.) according to the manufacturer's protocol. Briefly, cells were transfected with 1 µg lentiviral vector-green fluorescent protein (pLV-GFP) or pLV-RANKL (Shanghai GenePharma Co., Ltd.) at 37˚C on a 10 cm culture plate. Vectors were collected from the supernatants on days 2 or 3 post-transfection. Cells were then transferred to fresh DMEM (Invitrogen; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS and incubated at 37˚C for 24 h with various concentrations of lentivirus (107, 108 and 109 transducing U/ml). Pure infected cells were selected for GFP using flow cytometry and the data were analyzed with the Guava EasyCyte™ 8 software (EMD Millipore). A total of 98% of cells were reportedly positive for GFP. A549/DPP cells were transfected using the same method. Cells selected using G418 (Sigma-Aldrich; Merck KGaA) were considered to exhibit lncRNA RANKL overexpression according to the manufacturer's protocol. Empty vector was used as a negative control (NC). The current study was approved by Cangzhou Central Hospital (Cangzhou, China).

Small interfering RNA (siRNA) transfection

A549/DDP cells were added to six-well plates at a density of 5x103 cells/well. Cells were then transfected with 50 nM siRNA targeting RANKL (si-lncRNA RANKL, 5'-GCGACCAAUGUCAGGUCAUTT-3') or control siRNA (si-Control, 5'-AUGACCUGACAUUGGUCACTT-3'; both from Shanghai GenePharma, Co. Ltd.). Briefly, 50 nM siRNA was dissolved in 250 µl Opti-MEM medium containing 10 µl Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Each sample was thoroughly mixed and cultured for 5 min at 20˚C prior to the supplementation of the complex (500 µl in each well) for 48 h at 37˚C.

RNA isolation and reverse transcription quantitative PCR (RT-qPCR)

TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) was used to extract total RNA from cells according to the manufacturer's protocol. RT was performed using a PrimeScript RT reagent kit (Takara Bio, Inc.) according to the manufacturer's instructions. qPCR was performed using a QuantiTect SYBR-Green PCR kit (Qiagen GmbH) on an ABI 7300 Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.). The PCR thermocycling conditions were as follows: Initial denaturation for 5 min at 95˚C; followed by 36 cycles of 10 sec at 95˚C, 10 sec at 58˚C and 20 sec at 72˚C. GAPDH was used as an internal control for qPCR amplification. The relative quantification of target gene was conducted by using the 2-∆∆Cq method (20). The following primer pairs were used for the qPCR: GAPDH forward, 5'-CAAAAGGGTCATCTCC-3' and reverse, 5'-CCCCAGCATCAAAGGTG-3'; RANKL forward, 5'-CAGAAGATGGCACTCACTGCA-3' and reverse, 5'-CACCATCGCTTTCTCTGCTCT-3'.

Cell Counting Kit-8 (CCK-8) assay

DDP sensitivity of A549/DPP and A549 cells was assessed using the CCK-8 assay (Dojindo Molecular Technologies, Inc.) according to the manufacturer's instructions. Briefly, cells were seeded onto 96-well plates at a density of 4x103 cells/well and supplemented with 0, 1, 5, 10, 20, or 40 µg/ml DDP (Sigma-Aldrich; Merck KGaA). After 48 h, 10 µl CCK-8 was added into the culture medium for 4 h at 37˚C. Optical density of the supernatant was read at 490 nm using a microplate spectrophotometer. Absorbances were normalized to the untreated control cultures which represented 100% viability. % Viability=Mean absorbance of sample/Mean absorbance of control x100.

Cell death assessment

A549/DDP cell death was evaluated using the Annexin-V/propidium iodide (PI) Apoptosis Detection kit (Nanjing Jiancheng Bioengineering Institute) according to the manufacturer's protocol. Briefly, cells were washed twice with cold PBS and added to 1 ml binding buffer (BioVision, Inc.). The suspension was divided into 100 µl aliquots (1x105 cells) in fresh tubes and 5 µl PI and 5 µl Annexin-V were added. Cell survival, cell death and early and late cell apoptosis were evaluated via flow cytometry (Guava EasyCyte™ 8; EMD Millipore). Fluorescence signals were analyzed using a flow cytometer. Data were analyzed using the Guava EasyCyte™ 8 software (EMD Millipore). All assays were performed at least thrice.

Transwell assay

Transfected cells were centrifuged at 1,000 x g for 10 min at 20˚C and resuspended (density, 2.0x105/ml) in serum-free DMEM. Transwell chambers (8.0 µm pore) in 24-well plates were coated with Matrigel at 37˚C for 6 h and 200 µl cell suspension and 600 µl DMEM were added to the top and bottom of the chambers. Then, cells were fixed in 4% paraformaldehyde for 15 min at 20˚C and incubated for 48 h at 37˚C, followed by staining with 10% crystal violet for 15 min at 20˚C. Adherent cells were carefully removed and penetrating cells were collected. Cells were monitored by Nikon Optical TE2000-S inverted fluorescence microscope (magnification, x200). At least 12 randomly selected fields per well were, counted with ImageJ version 7 software (National Institutes of Health).

Western blotting

Cell lysates were homogenized using a RIPA lysis buffer (Bio-Rad Laboratories, Inc.) and protein content was quantified using the Bradford protein assay (Bio-Rad Laboratories, Inc.) according to the manufacturer's protocol. Proteins (40 µg/lane) were isolated on 8-15% Tris-HCl polyacrylamide gels and transferred to PVDF membranes, which were blocked with TBST at 4˚C for 1 h. Membranes were incubated overnight with primary antibodies at 4˚C [anti-p27 (cat. no. 3686; 1:1,000 dilution), anti-p53 (cat. no. 2527; 1:1,000 dilution), anti-AKT (cat. no. 4691; 1:1,000 dilution), anti-PI3K (cat. no. 4249; 1:1,000 dilution), anti-signal transducer and activator of transcription 3 (stat3; cat. no. 12640; 1:1,000 dilution), anti-p21 (cat. no. 2947; 1:1,000 dilution), anti-p-AKT (cat. no. 4060; 1:1,000 dilution), anti-p-PI3K (cat. no. 17366; 1:1,000 dilution) and anti-p-stat3 (cat. no. 9145; 1:1,000 dilution)] and anti-β-actin (cat. no. 4970; 1:1,000 dilution) (all from Cell Signaling Technology, Inc.). Membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies (cat. no. 7074; 1:1,000 dilution) at 20˚C for 2 h. Immuno-reactive bands were detected by ECL plus detection reagent (Pierce; Thermo Fisher Scientific, Inc.) and analyzed with ImageQuant™ LAS 4000 imaging system (Cytiva). Protein levels were determined by normalization to the level of β-actin with ImageQuant™ LAS 4000 imaging system version 8 (Cytiva).

Statistical analysis

Data are presented as the mean ± standard error of the mean of three experiments. Differences were evaluated using two-tailed, unequal variances Student's t-tests or ANOVA followed by Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

Results

RANKL expression is upregulated in A549/DDP cells

The CCK-8 assay revealed that following DDP exposure, IC50 (half maximal inhibitory concentration) significantly decreased in A549 cells compared with A549/DDP cells (Fig. 1A and B). Furthermore, RANKL expression was significantly decreased in A549 cells compared with A549/DDP cells (Fig. 1C). RANKL expression was also significantly upregulated in A549 cells following exposure to various concentrations of DDP for 48 h (Fig. 1D). These results indicated that RANKL expression is upregulated in A549/DDP cells.

Figure 1

RANKL expression was upregulated in A549/DDP cells. (A) The effect of DDP on the inhibition rate of A549/DDP and A549 cells. (B) The IC50 of DDP increased in A549/DDP cells compared with A549 cells. (C) RANKL expression in A549/DDP and A549 cells was assessed via RT-qPCR. (D) A549 cells were incubated with various concentrations of DDP (0, 1, 5,10, 20 or 40 µg/ml) for 48 h. RANKL expression was assessed via RT-qPCR. GAPDH was used as the internal control for RT-qPCR. Data are presented as mean ± standard error of the mean from three independent experiments. *P<0.05; **P<0.01 vs. A549 or 0 µg/ml DPP). RANKL, receptor activator of nuclear factor-κ B ligand; DDP, cisplatin; IC50, half of the maximal inhibitory concentration; RT-qPCR, reverse transcription-quantitative PCR.

RANKL contributed to DDP resistance in A549/DPP cells

A549/DPP cells were stably transfected with lentiviruses to investigate the effects of RANKL overexpression on DDP sensitivity. RANKL expression levels significantly increased following transfection compared with controls (Fig. 2A). Furthermore, RANKL overexpression significantly increased the IC50 of DDP (Fig. 2B and C). To further understand the role of RANKL in DDP resistance, A549/DDP cells were transfected with si-lncRNA RANKL or si-Control. Cells transfected with si-RANKL demonstrated significant RANKL knockdown compared with controls at 48 h post-transfection (Fig. 2D). The CCK-8 assay revealed that the IC50 of DDP was decreased by si-RANKL transfection in A549/DDP cells (Fig. 2E and F). Additionally, RANKL overexpression significantly increased the IC50 of DDP (Fig. 2G and H) in A549 cells.

Figure 2

RANKL contributes to DDP resistance in A549/DDP cells. (A) RANKL expression in A549/DPP cells that underwent stable transfection with RANKL lentiviruses was assessed using RT-qPCR. GAPDH was used as the internal control. (B) The effect of DDP on the inhibition rate of A549/DDP cells was evaluated via CCK-8 assay. (C) IC50 of DDP in A549/DPP cells was evaluated via CCK-8 assay. (D) Efficacy of siRNA in A549/DDP cells was assessed via RT-qPCR following 48 h transfection with si-Control or si-lncRNA RANKL. GAPDH was used as the internal control. (E) The effect of DDP on the inhibition rate of A549/DDP cells was evaluated via CCK-8 assay. (F) IC50 of DDP in A549/DDP cells was evaluated using the CCK-8 assay. (G) IC50 of DDP in A549 cells was evaluated using the CCK-8 assay. (H) The effect of DDP on the inhibition rate of A549 cells was evaluated using the CCK-8 assay. Data are presented as mean ± standard error of the mean. *P<0.05. **P<0.01. RANKL, receptor activator of nuclear factor-κ B ligand; DDP, cisplatin; RT-qPCR, reverse transcription-quantitative PCR; CCK-8, Cell Counting kit-8; IC50, half of the maximal inhibitory concentration; lncRNA, long non-coding RNA; si, short interfering.

RANKL decreases A549/DPP cell apoptosis

An apoptosis assay demonstrated that the overexpression of RANKL resulted in a decreased apoptosis rate in A549/DPP cells (Fig. 3A and B). In contrast, the downregulation of RANKL expression resulted in an increased apoptosis rate in A549/DPP cells (Fig. 3C and D). The results indicated that knockdown of RANKL expression is associated with increased apoptosis rate, thus reversing DDP resistance of A549/DDP cells. Additionally, RANKL overexpression significantly decreased apoptosis rate in A549 cells (Fig. 3E and F).

Figure 3

RANKL suppresses A549/DDP cell apoptosis. Flow cytometry was used to assess the apoptosis of A549/DDP cells transfected with a (A) control vector, (B) lncRNA RANKL, (C) si-Control, (D) si-lncRNA RANKL, (E) control vector or (F) lncRNA RANKL. Data are presented as mean ± standard error of the mean. *P<0.05; **P<0.01. RANKL, receptor activator of nuclear factor-κ B ligand; DDP, cisplatin; lncRNA, long non-coding RNA; si, short interfering; IC50, half of the maximal inhibitory concentration.

RANKL promotes migration of A549/DPP cells

Subsequently, transwell assays were conducted to investigate the migration ability of A549/DPP cells following RANKL overexpression and knockdown. The results demonstrated that the number of penetrating cells among A549/DPP cells transfected with RANKL was higher compared with controls, indicating enhanced migration ability (Fig. 4A and B). By contrast, the number of penetrating cells among A549/DPP cells transfected with si-RANKL was lower compared with controls, indicating inhibition of migration ability (Fig. 4C and D). Additionally, RANKL overexpression significantly promoted the migration ability of A549 cells (Fig. 4E and F).

Figure 4

RANKL regulates the migration of A549 and A549/DDP cells. (A) A549/DPP cells transfected with lncRNA RANKL exhibited remarkably increased migration ability. (B) Quantification of data shown in (A). (C) A549/DPP cells transfected with si-lncRNA-RANKL exhibited remarkably suppressed migration ability. (D) Quantification of data shown in (C). (E) A549 cells transfected with lncRNA RANKL exhibited significantly increased migration ability. (F) Quantification of data shown in (E). Data are presented as mean ± standard error of the mean. *P<0.05. RANKL, receptor activator of nuclear factor-κ B ligand; DPP, cisplatin; si, short interfering; lncRNA, long non-coding RNA.

RANKL knockdown activates p53 in A549/DPP cells

p53 is associated with cell apoptosis and resistance to antitumor drugs in various types of cancer (21). Therefore, whether RANKL regulates p53 expression was investigated. Western blotting demonstrated that the expressions of p53, p21 and p27 were upregulated following RANKL knockdown in A549/DPP cells (Fig. 5A-D), whereas these were downregulated after RANKL overexpression in A549/DPP cells (Fig. 5E-H).

Figure 5

RANKL knockdown stimulates p53, p21 and p27 expression in A549/DPP cells. (A) Representative immunoblots for p53, p21 and p27 expression and quantitative assessments of the concentration of (B) p53, (C) p21 and (D) p27 in A549/DPP cells following transfection with si-Control or si-lncRNA RANKL for 48 h. (E) Representative immunoblots for p53, p21 and p27 and quantitative assessments of the concentration of (F) p53, (G) p21 and (H) p27 in A549/DPP cells following temporary transfection with a si-Control or lncRNA RANKL for 24 h. Data are presented as mean ± standard error of the mean. **P<0.01. RANKL, receptor activator of nuclear factor-κ B ligand; DPP, cisplatin; si, short interfering; lncRNA, long non-coding RNA; NC, negative control.

RANKL knockdown inhibits the PI3K/AKT pathway in A549/DPP cells

The PI3K/AKT pathway has been confirmed to regulate cell functions including cell migration and proliferation (21). Therefore, whether RANKL regulates NSCLC development via this pathway was determined. Western blotting results demonstrated that the phosphorylated protein levels of PI3K, AKT and stat3 were downregulated following RANKL knockdown in A549/DPP cells (Fig. 6A-D), whereas levels were upregulated following RANKL overexpression in A549/DPP cells (Fig. 6E-H).

Figure 6

RANKL knockdown suppresses the PI3K/AKT pathway in A549/DPP cells. (A) Representative immunoblots for (A) p-PI3K, PI3K, p-AKT, AKT, p-stat3 and stat3 and quantitative assessments of the concentration of (B) p-PI3K/PI3K, (C) p-AKT/AKT and (D) p-stat3/stat3 in A549/DPP cells following temporary transfection with si-Control or si-lncRNA RANKL for 24 h. (E) Representative immunoblots for (A) p-PI3K, PI3K, p-AKT, AKT, p-stat3 and stat3 and quantitative assessments of the concentration of (F) p-PI3K/PI3K, (G) p-AKT/AKT and (H) p-stat3/stat3 (H) in A549/DPP cells following temporary transfection with a NC or lncRNA RANKL for 24 h. Data are presented as mean ± standard error of the mean. *P<0.05, **P<0.01. RANKL, receptor activator of nuclear factor-κ B ligand; p-, phosphorylated; PI3K, phosphatidylinositol 3-kinase; AKT, protein kinase B; DPP, cisplatin; stat3, signal transducer and activator of transcription 3; si, short interfering; lncRNA, long non-coding RNA; NC, negative control.

Discussion

Recent studies have demonstrated that p53 downregulation inhibits cell death and is associated with treatment resistance in various types of cancers, such as liver cancer (22-24). Reportedly, p53 stimulation triggered cell death following DDP exposure (25). The present study demonstrated that RANKL knockdown increased the expressions of p53, p21 and p27 in A549/DPP cells. It is well-known that p53 is upstream of p21 and p27(25). The results of the present study indicated that RANKL regulated apoptosis, migration and chemoresistance of NSCLC cells via p53 downregulation.

The PI3K/AKT pathway is important in tumorigenesis (26-28). PI3K/AKT exerts antiapoptotic effects primarily through its effects on numerous effector molecules, such as TGF and Bcl-2 (29,30). Apoptosis promotion has become a focus of tumor treatment and the PI3K/AKT pathway is believed to be important for developing new therapeutic targets for tumor cell metastasis (31). The present study demonstrated that PI3K, AKT and stat3 were remarkably downregulated following RANKL knockdown in A549/DPP cells, indicating that RANKL promotes the apoptosis and migration of NSCLC cells via the PI3K/AKT pathway.

In summary, the current study demonstrated that RANKL contributes to the DDP resistance of A549 cells. Additionally, RANKL suppressed p53 expression and enhanced the PI3K/AKT pathway, indicating that RANKL may serve as a promising therapeutic target for NSCLC. However, the present study did not study the RANKL-p53/PI3K/AKT pathway in animal models and patients. A subsequent study will investigate the RANKL in vivo in animal models and patients with NSCLC.