RNA interference screen to identify pathways that enhance or reduce nonviral gene transfer during lipofection

Abstract

Some barriers to DNA lipofection are well characterized; however, there is as yet no method of finding unknown pathways that impact the process. A druggable genome small-interfering RNA (siRNA) screen against 5,520 genes was tested for its effect on lipofection of human aortic endothelial cells (HAECs). We found 130 gene targets which, when silenced by pooled siRNAs (three siRNAs per gene), resulted in enhanced luminescence after lipofection (86 gene targets showed reduced expression). In confirmation tests with single siRNAs, 18 of the 130 hits showed enhanced lipofection with two or more individual siRNAs in the absence of cytotoxicity. Of these confirmed gene targets, we identified five leading candidates, two of which are isoforms of the regulatory subunit of protein phosphatase 2A (PP2A). The best candidate siRNA targeted the PPP2R2C gene and produced a 65% increase in luminescence from lipofection, with a quantitative PCR-validated knockdown of approximately 76%. Flow cytometric analysis confirmed that the silencing of the PPP2R2C gene resulted in an improvement of 10% in transfection efficiency, thereby demonstrating an increase in the number of transfected cells. These results show that an RNA interference (RNAi) high-throughput screen (HTS) can be applied to nonviral gene transfer. We have also demonstrated that siRNAs can be co-delivered with lipofected DNA to increase the transfection efficiency in vitro.

Figure 1. Human aortic endothelial cells were reverse transfected with sham control, negative control small-interfering RNA (siRNA), one of three siRNAs targeting the Renilla luciferase gene, or a pool of the three Renilla-targeting siRNAs

Two commercial transfection reagents, siPort NeoFX and RNAiFect, were used for reverse transfection. After siRNA reverse transfection and the subsequent lipofection of a Renilla luciferase plasmid DNA, luminescence from transfection and from viability were measured in each well, and from these data normalized luminescence was calculated. Each bar represents the mean-normalized luminescence, and the error bars represent 1 SD (n = 32). Significant changes with respect to the negative control (Neg) were determined using the Mann–Whitney U-test (*P ≤ 0.0001). Ren, Renilla-targeted control; RLU, relative light unit; RNAi, RNA interference.

Figure 2. An RNA interference high-throughput screen was performed on 5,520 genes with three small-intefering RNAs pooled per gene in 384-well plates

Two replicates of each gene were screened in independent plates, and the robust Z score was calculated for each. Knockdowns resulting in a robust Z score of 2 or greater or −2 or less in both plates were identified as positive and negative hits, respectively, identified by the red dots in the figure.

Figure 3. After the primary high-throughput screen, the three small- interfering RNAs (siRNAs) targeting the top positive hits were screened individually at 30 nmol/l

Luminescence data from (a) transfection and (b) viability are shown for the siRNAs corresponding to the five leading candidate genes, the negative control (Neg), and the Renilla-targeted control (Ren). Each bar represents the mean value, and error bars represent 1 SD (n = 4). Significant increases in transfection with respect to the negative control were calculated using the Mann–Whitney U-test (*P ≤ 0.05 and **P ≤ 0.01). RLU, relative light unit.

Figure 4. Lead candidates from the primary and confirmatory studies were screened in 96-well plates

(a) Raw transfection signal and (b) cell viability are shown after reverse transfection of small-interfering RNAs at 10 and 30 nmol/l with conditions scaled from the 384-well experiments. Each bar represents the mean value, and error bars represent 1 SD (n = 4). Significant increases in luminescence from lipofection with respect to the negative control were calculated using the Mann–Whitney U-test (*P ≤ 0.03). Neg, negative control; RLU, relative light unit.

Figure 5. Human aortic endothelial cells were seeded 1 day prior to co-transfection with small-interfering RNAs (siRNAs) and green fluorescent protein (GFP) plasmid DNA using Lipofectamine 2000 (Lipo2000)

The siRNAs co-delivered with plasmid were: (a) no siRNA; (b) GFP-targeting siRNA, siGFP; (c) negative control siRNA; (d) siRNA 104939; (e) siRNA 19286; and (f) siRNAs 104939 and 19286. Twenty-four hours after transfection, fluorescence microscopy pictures were taken (a–f). (g) The cells were subjected to flow cytometry analysis.

Figure 6. For quantitative real-time analysis, small-interfering RNAs (siRNAs) were reverse transfected into human aortic endothelial cells in 24-well plates, and RNA was harvested 24 hours later

The total RNA was reverse transcribed, and the PPP2R2C transcript was quantified for relative gene expression to verfiy the knockdown of the PPP2R2C gene. Each bar represents the mean value, and error bars represent 1 SD (n = 4 for all except for siRNA 104939, n = 2).