lncRNA‑u50535 promotes the progression of lung cancer by activating CCL20/ERK signaling

Abstract

The ligand/receptor pair C‑C motif chemokine ligand 20 (CCL20)/C‑C motif chemokine receptor 6 (CCR6) is considered to be highly activated in lung cancer and significantly accelerates lung cancer progression through activation of ERK signaling. In addition, it has been shown that long non‑coding RNA‑u50535 (lncRNA‑u50535) upregulates CCL20 expression and facilitates cancer progression in colorectal cancer (CRC). However, the effects of lncRNA‑u50535 in lung cancer progression and whether lncRNA‑u50535 regulates CCL20/CCR6/ERK signaling in lung cancer remain ill‑defined. Therefore, the aim of the present study was to investigate the effects of lncRNA‑u50535 on CCL20/CCR6/ERK signaling in lung cancer progression. The results demonstrated that lncRNA‑u50535 expression was upregulated in lung cancer tissues and cell lines compared with normal tissues and cells. Knockdown of lncRNA‑u50535 decreased lung cancer cell proliferation and migration, induced G0/G1 phase arrest and promoted cell apoptosis. Western blot and luciferase reporter gene assays demonstrated that lncRNA‑u50535 overexpression increased the translation and transcription of CCL20. In addition, knockdown of lncRNA‑u50535 decreased CCL20, CCR6 and p‑ERK levels. The effects of lncRNA‑u50535 on cell proliferation and cell apoptosis were weakened when CCL20 was silenced. Overall, the present study demonstrated that lncRNA‑u50535 may function as an oncogene in lung cancer progression by regulating CCL20/ERK signaling.

Figure 1.

Expression of lncRNA-u50535 in lung cancer tissues and cell lines. (A) LncRNA-u50535 expression levels in 20 paired lung cancer tissues and adjacent normal lung tissues. (B) Western blot analysis of the protein expression levels of CCL20 and CCR6 in lung cancer tissues and normal tissues. (C) lncRNA-u50535 expression levels in the normal lung cell line HBE and the lung cancer cell lines A549, H1299 and SPC-A1. (D) Western blot analysis of the protein expression levels of CCL20 and CCR6 in the normal lung cell line HBE and the lung cancer cell lines A549, H1299 and SPC-A1. *P<0.05, **P<0.01, compared with normal. lnc, long non-coding; CCL20, C-C motif chemokine ligand 20; CCR6, C-C motif chemokine receptor 6.

Figure 2.

Effects of lncRNA-u50535 overexpression and knockdown in lung cancer cells. A549 and H1299 cells were infected with OE-NC, OE-lncRNA-u50535, sh-NC or sh-lncRNA-u50535. (A) lncRNA-u50535 levels were examined by quantitative PCR, to confirm the efficiency of knockdown and overexpression. (B) Cell viability was determined by CCK-8 assay in A549 and (C) H1299 cells. (D) Cell cycle phase distribution was determined by flow cytometry. *P<0.05, compared with sh-NC; ^#P<0.05, compared with OE-NC (n=3). lnc, long non-coding; OE, overexpression; NC, negative control; sh, short hairpin RNA. Effects of lncRNA-u50535 overexpression and knockdown in lung cancer cells. (E) Apoptosis rates were evaluated by flow cytometry. (F) Cell invasion was assessed using Transwell chambers coated with Matrigel. *P<0.05, compared with sh-NC; ^#P<0.05, compared with OE-NC (n=3). lnc, long non-coding; OE, overexpression; NC, negative control; sh, short hairpin RNA

Figure 2.

Effects of lncRNA-u50535 overexpression and knockdown in lung cancer cells. A549 and H1299 cells were infected with OE-NC, OE-lncRNA-u50535, sh-NC or sh-lncRNA-u50535. (A) lncRNA-u50535 levels were examined by quantitative PCR, to confirm the efficiency of knockdown and overexpression. (B) Cell viability was determined by CCK-8 assay in A549 and (C) H1299 cells. (D) Cell cycle phase distribution was determined by flow cytometry. *P<0.05, compared with sh-NC; ^#P<0.05, compared with OE-NC (n=3). lnc, long non-coding; OE, overexpression; NC, negative control; sh, short hairpin RNA. Effects of lncRNA-u50535 overexpression and knockdown in lung cancer cells. (E) Apoptosis rates were evaluated by flow cytometry. (F) Cell invasion was assessed using Transwell chambers coated with Matrigel. *P<0.05, compared with sh-NC; ^#P<0.05, compared with OE-NC (n=3). lnc, long non-coding; OE, overexpression; NC, negative control; sh, short hairpin RNA

Figure 3.

lncRNA-u50535 overexpression upregulates CCL20 in A549 and H1299 cells. (A) Western blot analysis of CCL20 protein expression levels in A549 and H1299 cells infected with OE-NC, OE-lncRNA-u50535, sh-NC or sh-lncRNA-u50535 for 48 h. *P<0.05, compared with sh-NC; ^#P<0.05, compared with OE-NC (n=3). (B) A luciferase reporter gene assay was performed to assess the transcriptional activity of CCL20 in A549 and H1299 cells infected with OE-NC or OE-lncRNA-u50535 for 48 h. *P<0.05, compared with OE-NC (n=3). Lnc, long non-coding; CCL20, C-C motif chemokine ligand 20; OE, overexpression; NC, negative control; sh, short hairpin RNA.

Figure 4.

lncRNA-u50535 promotes the malignant phenotype of A549 and H1299 cells by activating the CCL20/CCR6/ERK signaling pathway. (A) CCL20 mRNA expression levels and (B) CCL20 protein expression levels were detected in order to confirm the knockdown efficiency. *P<0.05, compared with sh-NC. (C) A549 and H1299 cells were treated with OE-lncRNA-u50535, sh-CCL20, or sh-OE-lncRNA-u50535 combined with sh-CCL20, for 48 h. The protein expression patterns of CCR6 were determined by western blotting. (D) ERK and p-ERK expression levels were also examined by western blotting. *P<0.05, compared with control (infected with both OE-NC and sh-NC); ^#P<0.05, compared with OE-lncRNA-u50535 (n=3). lnc, long non-coding; CCL20, C-C motif chemokine ligand 20; CCR6, C-C motif chemokine receptor 6; sh, short hairpin RNA; NC, negative control; OE, overexpression; p-, phosphorylated. lncRNA-u50535 promotes the malignant phenotype of A549 and H1299 cells by activating the CCL20/CCR6/ERK signaling pathway. (E and F) Cell proliferation was assessed by CCK-8 assay. (G) Cell cycle phase distribution. *P<0.05, compared with control (infected with both OE-NC and sh-NC); ^#P<0.05, compared with OE-lncRNA-u50535 (n=3). lnc, long non-coding; CCL20, C-C motif chemokine ligand 20; CCR6, C-C motif chemokine receptor 6; sh, short hairpin RNA; NC, negative control; OE, overexpression; p-, phosphorylated. lncRNA-u50535 promotes the malignant phenotype of A549 and H1299 cells by activating the CCL20/CCR6/ERK signaling pathway. (H) Apoptosis rates were determined by flow cytometry. (I) Cell invasion was assessed by Transwell chambers coated with Matrigel. *P<0.05, compared with control (infected with both OE-NC and sh-NC); ^#P<0.05, compared with OE-lncRNA-u50535 (n=3). lnc, long non-coding; CCL20, C-C motif chemokine ligand 20; CCR6, C-C motif chemokine receptor 6; sh, short hairpin RNA; NC, negative control; OE, overexpression; p-, phosphorylated.

Figure 4.

lncRNA-u50535 promotes the malignant phenotype of A549 and H1299 cells by activating the CCL20/CCR6/ERK signaling pathway. (A) CCL20 mRNA expression levels and (B) CCL20 protein expression levels were detected in order to confirm the knockdown efficiency. *P<0.05, compared with sh-NC. (C) A549 and H1299 cells were treated with OE-lncRNA-u50535, sh-CCL20, or sh-OE-lncRNA-u50535 combined with sh-CCL20, for 48 h. The protein expression patterns of CCR6 were determined by western blotting. (D) ERK and p-ERK expression levels were also examined by western blotting. *P<0.05, compared with control (infected with both OE-NC and sh-NC); ^#P<0.05, compared with OE-lncRNA-u50535 (n=3). lnc, long non-coding; CCL20, C-C motif chemokine ligand 20; CCR6, C-C motif chemokine receptor 6; sh, short hairpin RNA; NC, negative control; OE, overexpression; p-, phosphorylated. lncRNA-u50535 promotes the malignant phenotype of A549 and H1299 cells by activating the CCL20/CCR6/ERK signaling pathway. (E and F) Cell proliferation was assessed by CCK-8 assay. (G) Cell cycle phase distribution. *P<0.05, compared with control (infected with both OE-NC and sh-NC); ^#P<0.05, compared with OE-lncRNA-u50535 (n=3). lnc, long non-coding; CCL20, C-C motif chemokine ligand 20; CCR6, C-C motif chemokine receptor 6; sh, short hairpin RNA; NC, negative control; OE, overexpression; p-, phosphorylated. lncRNA-u50535 promotes the malignant phenotype of A549 and H1299 cells by activating the CCL20/CCR6/ERK signaling pathway. (H) Apoptosis rates were determined by flow cytometry. (I) Cell invasion was assessed by Transwell chambers coated with Matrigel. *P<0.05, compared with control (infected with both OE-NC and sh-NC); ^#P<0.05, compared with OE-lncRNA-u50535 (n=3). lnc, long non-coding; CCL20, C-C motif chemokine ligand 20; CCR6, C-C motif chemokine receptor 6; sh, short hairpin RNA; NC, negative control; OE, overexpression; p-, phosphorylated.

Figure 4.

lncRNA-u50535 promotes the malignant phenotype of A549 and H1299 cells by activating the CCL20/CCR6/ERK signaling pathway. (A) CCL20 mRNA expression levels and (B) CCL20 protein expression levels were detected in order to confirm the knockdown efficiency. *P<0.05, compared with sh-NC. (C) A549 and H1299 cells were treated with OE-lncRNA-u50535, sh-CCL20, or sh-OE-lncRNA-u50535 combined with sh-CCL20, for 48 h. The protein expression patterns of CCR6 were determined by western blotting. (D) ERK and p-ERK expression levels were also examined by western blotting. *P<0.05, compared with control (infected with both OE-NC and sh-NC); ^#P<0.05, compared with OE-lncRNA-u50535 (n=3). lnc, long non-coding; CCL20, C-C motif chemokine ligand 20; CCR6, C-C motif chemokine receptor 6; sh, short hairpin RNA; NC, negative control; OE, overexpression; p-, phosphorylated. lncRNA-u50535 promotes the malignant phenotype of A549 and H1299 cells by activating the CCL20/CCR6/ERK signaling pathway. (E and F) Cell proliferation was assessed by CCK-8 assay. (G) Cell cycle phase distribution. *P<0.05, compared with control (infected with both OE-NC and sh-NC); ^#P<0.05, compared with OE-lncRNA-u50535 (n=3). lnc, long non-coding; CCL20, C-C motif chemokine ligand 20; CCR6, C-C motif chemokine receptor 6; sh, short hairpin RNA; NC, negative control; OE, overexpression; p-, phosphorylated. lncRNA-u50535 promotes the malignant phenotype of A549 and H1299 cells by activating the CCL20/CCR6/ERK signaling pathway. (H) Apoptosis rates were determined by flow cytometry. (I) Cell invasion was assessed by Transwell chambers coated with Matrigel. *P<0.05, compared with control (infected with both OE-NC and sh-NC); ^#P<0.05, compared with OE-lncRNA-u50535 (n=3). lnc, long non-coding; CCL20, C-C motif chemokine ligand 20; CCR6, C-C motif chemokine receptor 6; sh, short hairpin RNA; NC, negative control; OE, overexpression; p-, phosphorylated.

Figure 5.

lncRNA-u50535 overexpression enhances the tumor formation ability of lung cancer cells in vivo. A549 cells were stably infected with OE-NC, OE-lncRNA-u50535, sh-NC or sh-lncRNA-u50535, and then the stably infected cell lines were injected into nude mice to test the tumorigenesis of the cells. (A) Tumor weights are shown relative to the sh-NC group (n=6 mice per group). **P<0.01, compared with sh-NC; ^#P<0.05, compared with OE-NC. (B) Schematic of experimental design. lnc, long non-coding; OE, overexpression; NC, negative control; sh, short hairpin RNA.