Liposomal Spherical Nucleic Acids for Regulating Long Noncoding RNAs in the Nucleus

Abstract

Emerging evidence indicates that long noncoding RNAs (lncRNAs) are actively involved in a number of developmental and tumorigenic processes. Here, the authors describe the first successful use of spherical nucleic acids as an effective nanoparticle platform for regulating lncRNAs in cells; specifically, for the targeted knockdown of the nuclear-retained metastasis associated lung adenocarcinoma transcript 1 (Malat1), a key oncogenic lncRNA involved in metastasis of several cancers. Utilizing the liposomal spherical nucleic acid (LSNA) constructs, the authors first explored the delivery of antisense oligonucleotides to the nucleus. A dose-dependent inhibition of Malat1 upon LSNA treatment as well as the consequent up-regulation of tumor suppressor messenger RNA associated with Malat1 knockdown are shown. These findings reveal the biologic and therapeutic potential of a LSNA-based antisense strategy in targeting disease-associated, nuclear-retained lncRNAs.

Keywords: DNA nanoparticles; gene regulation; liposomal SNA; nuclear targeting; spherical nucleic acids.

Figure 1

(A) Representative images of Cy5-labeled LSNA uptake within the A549 cells. The DAPI and MFI intensity was measured near the center of the nucleus (yellow circle) and was used to examine the MFI through the cell in the Z-direction. (B) Mean fluorescence intensity for Cy5 at the center of the nucleus in A549 cells; *** - p<0.001; * - p<0.05; scale bar = 20 μm.

Figure 2

(A) Fluorescence in situ hybridization was used to examine expression of Malat1 and confirm knockdown within the nucleus. Scale bar = 20 μm. (B) The mean fluorescence intensity (MFI) of Malat1 was quantified to examine the reduction in Malat1 expression with Malat1-PS-LSNA treatment. A significant decrease in MFI is seen with Malat1-PS-LSNAs; *** - p<0.001.

Figure 3

Malat1 mRNA expression levels 48 h after treatment with Malat1-PS-LSNAs. (A) A concentration-dependent down regulation of Malat1 is seen with treatment. Free PS ASOs delivered with Lipofectamine RNAiMax at 0.1 μM were used as a positive (+) control. (B) Malat1-PS-LSNAs were used to treat A549 cells following a siRNA treatment to knockdown the nuclear trafficking protein RAN. A significant decrease in Malat1 gene knockdown was observed with RAN protein knockdown while no difference was observed with non-targeting (Scr) siRNA treatment; *** - p<0.001.

Figure 4

A) IFIT2 mRNA expression with treatment of Malat1-PS-LSNAs. A significant up-regulation was seen with Malat1-PS-LSNAs. (B) Cell viability was examined in A549 cells treated with PO and PS LSNAs (1 μM by DNA). Curcumin (50 μM), an apoptotic inducing agent, was used as a positive control (+). No change in viability was observed 24 hours after treatment; ** - p<0.01.

Scheme 1

The Cy5 mean fluorescence intensity (MFI) was quantified at the center of the nucleus in A549 cells. Z-stacks were generated with optical sections of 0.3 μm throughout the cell. When measuring the MFI intensity with the nucleus (yellow circle), we used the peaks in the DAPI MFI vs. Z-Displacement graph to identify the top and bottom of the nucleus (green line). The center of the nucleus was defined as the midpoint between these two edges (yellow line) where the Cy5 MFI was recorded.