Long Noncoding RNA DANCR Activates Wnt/β-Catenin Signaling through MiR-216a Inhibition in Non-Small Cell Lung Cancer

Abstract

Long noncoding RNA differentiation antagonizing nonprotein coding RNA (lncRNA-DANCR) is associated with poor prognosis in multiple cancers, and promotes cancer stemness and invasion. However, the exact mechanisms by which DANCR promotes non-small cell lung cancer (NSCLC) remain elusive. In this study, we determined that DANCR knockdown (KD) impeded cell migration and reduced stem-like characteristics in two NSCLC cell lines, A549 and H1755. Wnt signaling was shown to promote NSCLC proliferation, stemness, and invasion; therefore, we hypothesized that DANCR may regulate these activities through induction of the Wnt/β-catenin pathway. DANCR KD reduced β-catenin signaling and protein expression, and decreased the expression of β-catenin gene targets c-Myc and Axin2. One of the well-defined functions of lncRNAs is their ability to bind and inhibit microRNAs. Through in silico analysis, we identified tumor suppressor miR-216a as a potential binding partner to DANCR, and confirmed this binding through coimmunoprecipitation and luciferase-reporter assays. Furthermore, we show that DANCR-induced β-catenin protein expression may be blocked with miR-216a overexpression. Our findings illustrate a role of DANCR in NSCLC migration and stemness, and suggest a novel DANCR/miR-216a signaling axis in the Wnt/β-catenin pathway.

Keywords: Wnt; lncRNA; microRNA; non-small cell lung cancer; β-catenin.

Figure 1

LncRNA-DANCR is highly upregulated in NSCLC tumors and cell lines. (A) Analysis of The Cancer Genome Atlas data for DANCR expression in primary lung adenocarcinoma (n = 706) and squamous cell carcinoma (n = 626) compared to normal lung tissue (t test with Welch’s correction, **** p < 0.0001); (B) qRT-PCR measuring fold change of DANCR expression in NSCLC cell lines compared to lung epithelial cell line HBE2 normalized to 1 (n = 3, t test, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Figure 2

Knockdown (KD) of lncRNA-DANCR expression in NSCLC cells inhibits migration and clonogenic growth. (A) Fold change of DANCR expression in NSCLC cell lines A549, H358, H1975, and H1755 transiently transfected with two different DANCR siRNAs (n = 3, t test, ** p < 0.01, **** p < 0.0001); (B) effect of DANCR KD on cell growth of A549 and H1755 cells using Aqueous Cell Titer 96 reagent (n = 3, 2-way ANOVA, ** p < 0.01, *** p < 0.001); (C) cell impedance assay using xCELLigence RTCA SP system on A549 and H1755 DANCR KD cells compared to scramble control (n = 3); (D) colony-formation assay on A549 and H1755 two weeks after seeding (n = 3, t test, **** p < 0.0001).

Figure 3

Knockdown of lncRNA-DANCR expression in NSCLC cells inhibits stem-cell characteristics. (A) Fold change of SOX2 and ALDH gene expression in A549 DANCR KD cells compared to scramble control (n = 3, t test, *** p < 0.001), n = 3; t test, **** p < 0.0001); (B) fold change of Sox2 and Aldh1/2 protein expression in A549 DANCR KD cells compared to scramble control (n = 3; t test, ** p < 0.01, **** p < 0.0001); (C) tumorsphere formation in stable A549 DANCR KD cells compared to scramble control. Representative images of tumorspheres shown, scale bar = 100 γm (n = 3, t test, **** p < 0.0001).

Figure 3

Knockdown of lncRNA-DANCR expression in NSCLC cells inhibits stem-cell characteristics. (A) Fold change of SOX2 and ALDH gene expression in A549 DANCR KD cells compared to scramble control (n = 3, t test, *** p < 0.001), n = 3; t test, **** p < 0.0001); (B) fold change of Sox2 and Aldh1/2 protein expression in A549 DANCR KD cells compared to scramble control (n = 3; t test, ** p < 0.01, **** p < 0.0001); (C) tumorsphere formation in stable A549 DANCR KD cells compared to scramble control. Representative images of tumorspheres shown, scale bar = 100 γm (n = 3, t test, **** p < 0.0001).

Figure 4

LncRNA-DANCR regulates Wnt/β-catenin signaling. (A) β-Catenin activity in A549 cells cotransfected with TOPFlash luciferase reporter + DANCR shRNAs compared to cells transfected with TOPFlash luciferase reporter + scramble control (n = 3, t test, *** p < 0.001, **** p < 0.0001); (B) fold change of β-catenin protein expression in A549 DANCR KD cells compared to scramble control (n = 3, t test, * p < 0.05); (C) fold change of CMYC and AXIN2 gene expression in A549 DANCR KD cells compared to scramble control (n = 3, t test, * p < 0.05, ** p < 0.01, *** p < 0.001); (D) fold change of c-myc and Axin2 protein expression in A549 DANCR KD cells compared to scramble control (n = 3, t test, * p < 0.05, ** p < 0.01).

Figure 4

LncRNA-DANCR regulates Wnt/β-catenin signaling. (A) β-Catenin activity in A549 cells cotransfected with TOPFlash luciferase reporter + DANCR shRNAs compared to cells transfected with TOPFlash luciferase reporter + scramble control (n = 3, t test, *** p < 0.001, **** p < 0.0001); (B) fold change of β-catenin protein expression in A549 DANCR KD cells compared to scramble control (n = 3, t test, * p < 0.05); (C) fold change of CMYC and AXIN2 gene expression in A549 DANCR KD cells compared to scramble control (n = 3, t test, * p < 0.05, ** p < 0.01, *** p < 0.001); (D) fold change of c-myc and Axin2 protein expression in A549 DANCR KD cells compared to scramble control (n = 3, t test, * p < 0.05, ** p < 0.01).

Figure 5

LncRNA-DANCR is a competitive inhibitor of miR-216a. (A) Potential binding position of DANCR MRE and miR-216a; (B) (top) schematic of MCP-MS2 immunoprecipitation assay; (bottom) MCP–MS2 immunoprecipitation assay using A549 DANCR KD cells transfected with MS2-DANCR compared to MS2-control cells (n = 3, t test, *** p < 0.001); (C) (top) schematic of luciferase-DANCR reporter assay; (bottom) 293T cells cotransfected with luciferase-DANCR reporter + miR-216a expression plasmid compared to luciferase-DANCR reporter + vector control cells (n = 3, t test, *** p < 0.001); (D) fold change of miR-216a expression in NSCLC cell lines compared to lung epithelial cell line HBE2 (n = 3, t test, *** p < 0.001, **** p < 0.0001).

Figure 5

LncRNA-DANCR is a competitive inhibitor of miR-216a. (A) Potential binding position of DANCR MRE and miR-216a; (B) (top) schematic of MCP-MS2 immunoprecipitation assay; (bottom) MCP–MS2 immunoprecipitation assay using A549 DANCR KD cells transfected with MS2-DANCR compared to MS2-control cells (n = 3, t test, *** p < 0.001); (C) (top) schematic of luciferase-DANCR reporter assay; (bottom) 293T cells cotransfected with luciferase-DANCR reporter + miR-216a expression plasmid compared to luciferase-DANCR reporter + vector control cells (n = 3, t test, *** p < 0.001); (D) fold change of miR-216a expression in NSCLC cell lines compared to lung epithelial cell line HBE2 (n = 3, t test, *** p < 0.001, **** p < 0.0001).

Figure 6

DANCR promotes Wnt/β-catenin signaling through miR-216a inhibition. (A) qRT-PCR measuring fold change of DANCR expression in A549 cells transfected with pBABE-DANCR compared to vector control (n = 3; t test, ** p < 0.001); (B) fold change of β-catenin protein expression in A549 DANCR overexpressing cells (n = 3, t test, ** p < 0.001); (C) (left) qRT-PCR measuring fold change of DANCR expression in A549 cells doubly transfected with vector + vector (EV + EV), vector + pBABE-miR-216a (EV + miR-216a), vector + pBABE-DANCR (EV + DANCR), or pBABE-DANCR + pBABE-miR-216a (DANCR + miR-216a); (right) qRT-PCR measuring fold change of miR-216a expression (n = 4, t test, * p < 0.05, ** p < 0.001); (D) fold change of β-catenin protein expression in A549 cells doubly transfected with vector + vector (EV + EV), vector + pBABE-miR-216a (EV + miR-216a), vector + pBABE-DANCR (EV + DANCR), or pBABE-DANCR + pBABE-miR-216a (DANCR + miR-216a) (n = 4, t test, * p < 0.05).

Figure 6

DANCR promotes Wnt/β-catenin signaling through miR-216a inhibition. (A) qRT-PCR measuring fold change of DANCR expression in A549 cells transfected with pBABE-DANCR compared to vector control (n = 3; t test, ** p < 0.001); (B) fold change of β-catenin protein expression in A549 DANCR overexpressing cells (n = 3, t test, ** p < 0.001); (C) (left) qRT-PCR measuring fold change of DANCR expression in A549 cells doubly transfected with vector + vector (EV + EV), vector + pBABE-miR-216a (EV + miR-216a), vector + pBABE-DANCR (EV + DANCR), or pBABE-DANCR + pBABE-miR-216a (DANCR + miR-216a); (right) qRT-PCR measuring fold change of miR-216a expression (n = 4, t test, * p < 0.05, ** p < 0.001); (D) fold change of β-catenin protein expression in A549 cells doubly transfected with vector + vector (EV + EV), vector + pBABE-miR-216a (EV + miR-216a), vector + pBABE-DANCR (EV + DANCR), or pBABE-DANCR + pBABE-miR-216a (DANCR + miR-216a) (n = 4, t test, * p < 0.05).