Regulating Oncogenic LncRNA DANCR with Targeted ECO/siRNA Nanoparticles for Non-Small Cell Lung Cancer Therapy

Abstract

Long noncoding RNA (lncRNA) differentiation antagonizing noncoding RNA (DANCR) is a proven oncogenic lncRNA across multiple cancer types. Its effects on cancer cell migration and invasion position it as a potential target for therapy on multiple levels of gene regulation. DANCR is overexpressed in non-small cell lung cancer (NSCLC), the most common lung cancer subtype with poor patient survival. To effectively deliver small interfering RNA (siRNA) against DANCR for NSCLC therapy, we used arginine-glycine-aspartic acid (RGD)-poly(ethylene glycol) (PEG)-(1-aminoethyl)-iminobis[N-oleicylcysteinyl-1-aminoethyl)propionamide] (ECO)/small interfering RNA against DANCR (siDANCR) nanoparticles to transfect A549 and NCI-H1299 cells. Over 90% DANCR silencing was observed along with inhibition of cell migration, invasion, and spheroid formation relative to transfection with negative control siRNA in RGD-PEG-ECO nanoparticles. DANCR knockdown further showed efficacy in reducing migration and invasion of epidermal growth factor receptor (EGFR)-inhibitor resistant NSCLC along with resensitization to the inhibitor. RGD-PEG-ECO/siDANCR demonstrated silencing for up to 7 d following a single transfection. The results suggest nanoparticle-mediated RNA interference against DANCR as a potential approach for NSCLC treatment by regulating cell migration and invasion in addition to improving EGFR inhibitor response.

Figure 1

Formation and characterization of RGD-PEG-ECO/siRNA nanoparticles. (A) Structure of ECO and scheme of RGD-PEG-ECO/siRNA formation. (B) Gel encapsulation assay shows strong entrapment of siDANCR and siNS in RGD-PEG-ECO nanoparticles with free siRNA as controls. RGD-PEG-ECO nanoparticles have uniform size (C) and zeta potential distributions (D) as measured with dynamic light scattering. The measured size is 119 nm for RGD-PEG-ECO/siDANCR nanoparticles and 116 nm for RGD-PEG-ECO/siNS nanoparticles; zeta potential is ca. +14 and +13 mV, respectively. (E) Confocal images (20× magnification) taken after 24 h blocking with excess free RGD peptide show reduced RGD-PEG-ECO nanoparticle uptake. Ratio of AF647 to Hoechst 33342 signal intensity is higher when no free RGD peptide is present in the transfection media (−RGD) compared to when excess RGD peptide is added (+RGD). Red: AF647; blue: Hoechst 33342 (n = 3). NC represents the negative control siRNA, siNS. siD represents siDANCR. Error bars denote standard error of measure; scale bars in all panels = 100 μm. ** p < 0.01, and *** p < 0.005 using unpaired t-test.

Figure 2

DANCR expression and downstream effects of silencing. (A) The NCI-H1299 cell line expresses DANCR mRNA at a 1.5× greater level relative to the A549 cell line as determined with a qRT-PCR analysis (n = 3). (B) Treatment with RGD-PEG-ECO/siDANCR nanoparticles silence DANCR expression over 90% relative to control (n = 3). (C) DANCR silencing in the NCI-H1299 cell line persists over the course of 6 d, with strongest silencing observed at 2 d post-transfection (n = 3). (D) Markers of epithelial-to-mesenchymal transition (EMT) are reduced on the mRNA level following DANCR silencing with ECO/siDANCR nanoparticles as shown with qRT-PCR (n = 3). (E) On the protein level, the same markers as in (D) similarly show reduced expression after silencing DANCR (n = 3). Quantification of western blot images shown as a ratio of the band of interest normalized to the loading control, β-actin. Error bars denote standard error of measure, * p < 0.05, ** p < 0.01, and *** p < 0.005 using unpaired t-test.

Figure 3

ECO/siDANCR nanoparticles inhibit lung cancer cell migration and invasion in Transwell assays. Across both A549 (A) and NCI-H1299 (C) cell lines (n = 3), cell migration (without Matrigel coating) and invasion (with Matrigel coating) are reduced upon silencing DANCR expression. Counting the number of stained cells migrating or invading across the Transwell membrane quantifies the reduction in migration and invasion for A549 (B) and NCI-H1299 (D) cells. Wound-healing assays revealed slower migration after DANCR knockdown in A549 (E) and NCI-H1299 (G) (n = 3). Quantification of wound-healing assay images as distance migrated relative to the original boundary positions over the course of the experiment for both A549 (F) and NCI-H1299 (H). (I) 3D growth on Matrigel after 5 d revealed smaller spheroid size after DANCR knockdown (n = 3). (J) Staining of the spheroids in (I) with ZD2-Cy5.5 (red) and Hoechst 33342 (blue) and subsequent confocal imaging showed the relative expression of EDB protein in the extracellular matrix. (K) Ratio of Cy5.5 maximum intensity to Hoechst maximum intensity in staining from (J) was reduced with DANCR silencing. Error bars denote the standard error of measure; scale bars in all panels = 100 μm. “NC” represents negative control RGD-PEG-ECO/siNS, “siD” represents RGD-PEG-ECO/siDANCR. * p < 0.05, ** p < 0.01, and *** p < 0.005 using unpaired t-test.

Figure 4

Drug-resistant NSCLC cells respond to DANCR silencing. (A) mRNA expression of the drug transporter MDR1, DANCR, and ECM protein EDB fibronectin are elevated in drug-resistant cells relative to parental cells (n = 3). (B) Cell viability relative to untreated control shows significant reduction in gefitinib IC₅₀ following transfection with RGD-PEG-ECO/siDANCR (n = 3). The IC₅₀ was initially 50 μM gefitinib for both A549-DR and NCI-H1299-DR but was reduced to 25 μM gefitinib for NCI-H1299-DR with RGD-PEG-ECO/siDANCR transfection. (C) Transwell migration and invasion assays (n = 3) show significant reduction in the number of migrated and invaded cells, respectively, following DANCR knockdown relative to transfection with control siRNA in both cell lines. (D) Quantification of number of cells migrated or invaded in A549 DR or NCI-H1299 DR cells from (C). (E) Smaller 3D spheroid growth was observed with DANCR silencing relative to control siRNA transfection (n = 3). Error bars denote standard error of measure, scale bars in all panels = 100 μm. * p < 0.05 and ** p < 0.01 using unpaired t-test.

Figure 5

DANCR silencing with RGD-PEG-ECO/siDANCR is sustained over time. (A) RGD-PEG-ECO/siDANCR nanoparticles showed a functional effect in reducing cell viability after transfection relative to RGD-PEG-ECO/siNS nanoparticles or untreated control groups in both NCI-H1299 and A549 cells (n = 3). Viability was expressed relative to untransfected cells. (B) A single 48 h transfection with RGD-PEG-ECO/siDANCR was sufficient to reduce DANCR expression in A549 and NCI-H1299 cells for 7 d relative to a single 48 h transfection with RGD-PEG-ECO/siNS (n = 3). (C) Cell migration and invasion (n = 3) assessed 7 d following the initial transfection performed in (B) showed a greater reduction in NCI-H1299 cells than in A549 cells, where DANCR expression recovered to 20% and 50%, respectively. (D) Quantification of the number of cells migrated or invaded in A549 DR or NCI-H1299 DR cells. (E) In drug-resistant NSCLC cells, a single transfection of RGD-PEG-ECO/siDANCR showed a reduction in DANCR expression lasting for 7 d and fully recovering to pretransfection level at 10 d (n = 3). Error bars denote standard error of measure, scale bars = 100 μm. * p < 0.05, ** p < 0.01, and *** p < 0.005 using unpaired t-test.