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

Abstract

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.

Keywords: cisplatin resistance; long non-coding RNA receptor activator of nuclear factor-κ B ligand; non-small-cell lung cancer; p53.

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.

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.

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.

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.

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.

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.