LncRNA DLX6-AS1 promotes the proliferation, invasion, and migration of non-small cell lung cancer cells by targeting the miR-27b-3p/ GSPT1 axis

Abstract

Background: Non-small cell lung cancer (NSCLC) has a significant impact on human health. The aim of this study was to explore the role of long non-coding RNA DLX6-AS1 in the proliferation, migration, and invasion of NSCLC cells. Methods: The expression of DLX6-AS1 in NSCLC tumor tissues and cell lines was examined by qRT-PCR. The effects of DLX6-AS1 knockdown on cell proliferation, migration, and invasion were assessed by Cell Counting Kit-8, wound healing, and transwell assays, respectively. Bioinformatics analyses, luciferase reporter assays, and RNA pull-down assays were employed to examine the mechanism by which DLX6-AS1 exerted its oncogenesis effects in NSCLC. The anti-tumor effect of silencing DLX6-AS1 in vivo was also evaluated. Results: DLX6-AS1 was over-expressed in NSCLC tumor tissues and cell lines and its level of expression was found to be associated with tumor size and advanced clinical stage in patients with NSCLC. Downregulation of DLX6-AS1 inhibited cell proliferation, cell clone formation, migration, and invasion of NSCLC cells. DLX6-AS1 was found to interact with miR-27b-3p/GSPT1. DLX6-AS1 expression was negatively correlated with miR-27b-3p expression, but positively correlated with GSPT1 expression in NSCLC samples. DLX6-AS1 knockdown also effectively suppressed tumor growth in an in vivo xenograft model. Conclusion: DLX6-AS1 regulated NSCLC progression by targeting the miR-27b-3p/GSPT1 axis, which may provide novel insights for NSCLC prognosis and therapy.

Keywords: DLX6-AS1; GSPT1; NSCLC; invasion; miR-27b-3p.

Figure 1

The expression of DLX6-AS1 was up-regulated in NSCLC samples and cell lines. (A) The DLX6-AS1 expression in 51 paired NSCLC clinical samples and normal tissues was detected by qRT-PCR. (B) The expression of DLX6-AS1 in NSCLC cell lines (CALU3, CALU6, A549, and H1299) and HBE cells were detected by qRT-PCR. Data are shown as the mean ± standard deviation. ***P<0.001 vs HBE cell line. Abbreviations: HBE, human bronchial epithelial; NSCLC, Non-small cell lung cancer.

Figure 2

DLX6-AS1 regulated cell proliferation, cloning formation, migration and invasion of NSCLC cells. (A) A549 and H1229 cells were transfected with pLKO.1-DLX6-AS1 or pLKO.1 plasmids, the transfection efficiency was evaluated by qRT-PCR analysis. (B) Cell proliferation of A549 and H1229 cells transfected with pLKO.1-DLX6-AS1 or pLKO.1 was identified by CCK8 assay. (C) Cell cloning capability of A549 and H1229 cells transfected with pLKO.1-DLX6-AS1 or pLKO.1 was examined by colony formation assay. (D) Cell migration of A549 and H1229 cells transfected with pLKO.1-DLX6-AS1 or pLKO.1 was examined by wound healing. (E) Cell migration of A549 and H1229 cells transfected with pLKO.1-DLX6-AS1 or pLKO.1 was examined by transwell assay. Data are shown as the mean ± standard deviation, ***P<0.001.

Figure 3

DLX6-AS1 could interact with miR-27b-3p/GSPT1. (A) miR-27b-3p binding sites in DLX6-AS1 were predicted by bioinformatics analysis. (B) Luciferase reporter assays were performed using HEK293T cells co-transfected with the miR-27b-3p mimic and DLX6-AS1-wt or DLX6-AS1-mut reporter plasmid. (C) The expression of miR-27b-3p in A549 and H1229 cells transfected with pLKO.1-DLX6-AS1 or pLKO.1 was examined by qRT-PCR. (D) The expression of miR-27b-3p in NSCLC tissues was identified, which showed negative correlation with DLX6-AS1 expression. (E) The expressions of miR-27b-3p in NSCLC cell lines and HBE cells were determined by qRT-PCR. (F) miR-27b-3p binding sites in GSPT1 were predicted by bioinformatics analysis. (G) Luciferase reporter assays were performed using HEK293T cells co-transfected with the miR-27b-3p mimic and GSPT1-wt or GSPT1-AS1-mut reporter plasmid. (H) The expression of GSPT1 in NSCLC tissues was identified, which showed negative correlation with miR-27b-3p expression. (I) The expression of GSPT1 in A549 and H1229 cells transfected with pLKO.1-DLX6-AS1 or pLKO.1 was examined by western blot analysis. ***P<0.01.

Figure 4

DLX6-AS1 knockdown inhibited cell proliferation, migration and invasion of NSCLC cells through targeting miR-27b-3p. A549 cells were transfected pLKO.1+NC mimics, pLKO.1-DLX6-AS1+NC mimics, pLKO.1-DLX6-AS1+ miR-27b-3p mimics. Cell proliferation (A), cloning formation (B), migration (C) and invasion (D) were identified by CCK8, cell cloning formation, wound healing and transwell assays, respectively. Data are shown as the mean ± standard deviation, ***P<0.001.

Figure 5

DLX6-AS1 promoted tumor growth in vivo. (A) Tumor growth curves were established by measuring tumor volume every 5 for 25 days after injection. (B) Tumor weights isolated from nude mice in each treatment group were determined on day 25 after injection. (C) GSPT1 and Ki67 expressions in tumor tissue were evaluated by IHC analysis. Data are shown as the mean ± standard deviation, ***P<0.001.