Long intergenic non‑coding RNA 00467 promotes lung adenocarcinoma proliferation, migration and invasion by binding with EZH2 and repressing HTRA3 expression

Abstract

Long non‑coding RNAs (lncRNAs) have been identified to serve an important role in the occurrence, development and metastasis of tumours. However, the role of linc00467 in lung adenocarcinoma (LAD) is unclear. In the present study, it was demonstrated that linc00467 expression was upregulated in human lung tumour tissues compared with normal tissues. In addition, high levels of linc00467 expression were associated with larger tumour sizes and later TNM stages. Functional experiments suggested that linc00467 promoted LAD cell proliferation, migration and invasion, and inhibited apoptosis in vitro. Knockdown of linc00467 altered the expression of downstream genes, including HtrA serine peptidase 3 (HTRA3), and RNA immunoprecipitation and chromatin immunoprecipitation assays indicated that linc00467 recruited EZH2 to the HTRA3 promoter to inhibit its expression. Taken together, the results of the present study indicated that linc00467 served an oncogenic role in LAD tumourigenesis, suggesting that it may be used as a novel diagnostic biomarker and therapeutic target for LAD.

Figure 1.

Bioinformatic analysis of the relative expression levels of linc00467 in LAD and normal tissues. (A-D) The relative expression of linc00467 was increased in LAD tissues compared with normal tissues based on the 4 Gene Expression Omnibus datasets: (A) GSE30219; (B) GSE19188; (C) GSE19804; and (D) GSE27262. **P<0.01. (E) The relative expression of linc00467 in LAD tissues compared with normal tissues was analysed using the TCGA database. TCGA data also indicated that linc00467 expression was upregulated in lung cancer tissues compared with normal tissues. LAD, lung adenocarcinoma; linc, long intergenic non-coding RNA; TCGA, The Cancer Genome Atlas.

Figure 2.

linc00467 is upregulated in LAD tissues. (A) linc00467 expression in LAD (n=60) compared with matched adjacent normal tissues (n=60) was examined by reverse transcription quantitative polymerase chain reaction, and linc00467 was clearly upregulated in lung cancer tissues. (B) linc00467 expression was increased in tumours with a maximum diameter >3 cm. (C) linc00467 expression was increased in stage III tumours. *P<0.05 and **P<0.01. linc, long intergenic non-coding RNA; LAD, lung adenocarcinoma.

Figure 3.

Abundance of linc00467 in LAD cells. (A) linc00467 expression was analysed by RT-qPCR in 3 LAD H1299, A549 and PC9 cell lines and in the normal bronchial epithelial 16HBE cell line. A549 and H1299 cells expressed increased levels of linc00467 compared with the 16HBE cells, while PC9 cells expressed relatively decreased linc00467 levels compared with the A549 and H1299 cells. (B-D) RT-qPCR was performed to detect the relative cytoplasmic and nuclear linc00467 levels in (B) A549, (C) H1299 and (D) PC9 cells. GAPDH was used as a cytoplasmic expression control, and U6 was used as a nuclear expression control. linc00467 was localized in of the nucleus and cytosol but was localized to a greater extent in the nucleus. (E and F) Relative expression of linc00467 in (E) A549 and (F) H1299 cells transfected with siRNAs. linc00467 was knocked down in A549 and H1299 cells by transfection with siRNAs. (G) Relative expression of linc00467 in PC9 cells transfected with the pcDNA3.1-linc00467 vector. linc00467 was overexpressed in PC9 cells via transfection with the pcDNA3.1-linc00467 vector. The data were obtained from at least 3 independent experiments and are presented as the mean ± standard deviation. *P<0.05 and **P<0.01. linc, long intergenic non-coding RNA; LAD, lung adenocarcinoma; RT-qPCR, reverse transcription quantitative polymerase chain reaction; siRNA; small interfering RNA; ns, not significant; NC, negative control.

Figure 4.

Effects of linc00467 on LAD cell proliferation in vitro. (A-C) MTT assays were performed to determine the viability of si-linc00467-transfected (A) A549 and (B) H1299 cells or (C) pcDNA3.1-linc00312-transfected PC9 cells. The MTT assays indicated that A549 and H1299 cell viability was decreased following knockdown of linc00467, while cell viability increased in pcDNA3.1-linc00467-treated PC9 cells. (D-F) Colony formation assays were conducted to determine the proliferative ability of transfected LAD cells. The colony formation ability of (D) A549 and (E) H1299 cells was significantly impaired following linc00467 knockdown, while (F) linc00467 overexpression increased PC9 cell colony formation ability. The data were obtained from at least 3 independent experiments and are presented as the means ± standard deviation. Magnification, ×100. *P<0.05 and **P<0.01. linc, long intergenic non-coding RNA; LAD, lung adenocarcinoma; siRNA, small interfering; NC, negative control.

Figure 5.

Effect of linc00467 on LAD apoptosis. Flow cytometry was used to analyse the apoptotic rates of si-linc00467-transfected A549 and H1299 cells and pcDNA3.1-linc00467-transfected PC9 cells. (A and B) Flow cytometric analyses indicated that linc00467 knockdown significantly increased apoptosis in (A) A549 and (B) H1299 cells. (C) linc00467 overexpression decreased PC9 apoptosis. The data were obtained from at least 3 independent experiments and are presented as the mean ± standard deviation. *P<0.05 and **P<0.01. linc, long intergenic non-coding RNA; LAD, lung adenocarcinoma; siRNA, small interfering; NC, negative control.

Figure 6.

Effect of linc00467 on LAD cell migration and invasion ability. Transwell assays were conducted to observe the migration and invasion ability of si-linc00467-transfected A549 and H1299 cells and pcDNA3.1-linc00467-transfected PC9 cells. (A and B) linc00467 knockdown significantly suppressed the migration and invasion ability of (A) A549 and (B) H1299 cells. (C) linc00467 overexpression increased the migration and invasion ability of PC9 cells. The data were obtained from at least 3 independent experiments and are presented as the mean ± standard deviation. Magnification, ×100. **P<0.01. linc, long intergenic non-coding RNA; LAD, lung adenocarcinoma; siRNA, small interfering RNA; NC, negative control.

Figure 7.

Relative expression of linc00467 in A549 and H1299 cells transfected with linc00467-shRNA. (A and B) linc00467 was knocked down in (A) A549 and (B) H1299 cells by transfection with linc00467-shRNA. **P<0.01. linc, long intergenic non-coding RNA; shRNA, short hairpin RNA.

Figure 8.

Gene expression profiling. (A and B) RNA transcriptome sequencing was used to analyse the gene expression profile in H1299 cells following linc00467 knockdown. A total of 625 differentially expressed transcripts were identified (471 upregulated transcripts and 154 downregulated transcripts, |log2 (fold change)|> 1 and P<0.05). A volcano plot and heat-map present all of the differentially expressed genes between linc00467-knockdown H1299 and control cells. (C and D) Reverse transcription quantitative polymerase chain reaction was used to verify the mRNA expression levels of 6 randomly selected genes in control shRNA vs. linc00467 shRNA-transfected (C) A549 and (D) H1299 cells. *P<0.05 and **P<0.01. linc, long intergenic non-coding RNA; shRNA, short hairpin RNA.

Figure 9.

linc00467 regulates the mRNA and protein levels of HTRA3. (A and B) Changes in HTRA3 mRNA and protein expression levels in (A) A549 and (B) H1299 cells transfected with linc00467-shRNA as determined by reverse transcription quantitative polymerase chain reaction. (C and D) Changes in HTRA3 mRNA and protein expression levels in (C) A549 and (D) H1299 cells transfected with linc00467-shRNA as determined by western blot analysis. HTRA3 exhibited increased mRNA and protein expression levels in linc00467-knockdown A549 cells and H1299 cells compared with in control cells. The data were obtained from at least 3 independent experiments and are presented as the means ± standard deviation. **P<0.01. linc, long intergenic non-coding RNA; HTRA3, HtrA serine peptidase 3; shRNA, short hairpin RNA.

Figure 10.

linc00467 represses HTRA3 expression by binding with EZH2. (A and B) EZH2 was knocked down in (A) A549 and (B) H1299 by transfection with siRNAs. The relative expression of EZH2 in siRNA-transfected A549 and H1299 cells is shown. (C and D) HTRA3 protein level changes in (C) A549 and (D) H1299 cells transfected with si-EZH2, as determined by western blot analysis. HTRA3 protein expression levels were increased in the EZH2-downregulated group compared with in the control group. (E and F) RNA immunoprecipitation experiments were performed in (E) A549 and (F) H1299 cells, and the coprecipitated RNA was subjected to RT-qPCR analysis for the presence of linc00467. The expression levels of linc00467 RNA are presented as the fold enrichment in EZH2 relative to the IgG immunoprecipitates. It was observed that linc00467 directly bound EZH2 in A549 and H1299 cells. (G) Chromatin immunoprecipitation RT-qPCR of EZH2 occupancy on the HTRA3 promoter in A549 and H1299 cells, with IgG as a negative control. The results indicated that EZH2 was able to bind the HTRA3 promoter. The data were obtained from at least 3 independent experiments and are presented as the mean ± standard deviation. *P<0.05 and **P<0.01. linc, long intergenic non-coding RNA; HTRA3, HtrA serine peptidase 3; EZH2, histone-lysine N-methyltransferase EZH2; siRNA, small interfering RNA; RT-qPCR, reverse transcription quantitative polymerase chain reaction; NC, negative control.

Figure 11.

HTRA3 inhibition attenuates the inhibitory effect on proliferation, migration and invasion of LAD cells induced by linc00467 depletion. (A and B) Cell proliferation, migration and invasion were measured in (A) A549 and (B) H1299 cells transfected with control shRNA, linc00467 shRNA and linc00467 shRNA+si-HTRA3. (C and D) Cell proliferation, migration and invasion were inhibited in linc00467-knockdown (C) A549 and (D) H1299 cells, whereas knockdown of HTRA3 partially reversed all of these inhibitory effects. The data were obtained from at least 3 independent experiments and are presented as the mean ± standard deviation. Magnification, ×100. *P<0.05 and **P<0.01. HTRA3, HtrA serine peptidase 3; linc, long intergenic non-coding RNA; shRNA, short hairpin RNA; siRNA, small interfering RNA; NC, negative control.

Figure 12.

Relative expression and clinical significance of HTRA3 expression in LAD tissues. (A) The expression level of HTRA3 in LAD tissues was decreased compared with that in adjacent normal lung tissues. (B-D) HTRA3 expression was decreased in (B) tumours with a maximum diameter >3 cm, (C) stage III tumours and (D) high lymph node metastasis.. *P<0.05 and **P<0.01. HTRA3, HtrA serine peptidase 3; LAD, lung adenocarcinoma; linc, long intergenic non-coding RNA.