Reagents

The antibiotic solution (containing 10,000 U/mL penicillin and 10 mg/mL streptomycin), the trypsin-EDTA mixture (containing 0.05% trypsin and EDTA), RPMI-1640, FBS (foetal bovine serum) and pHrodo™ Red succinimidyl ester were obtained from Invitrogen (Carlsbad, CA). The pairs of each small interfering RNA (siRNA) and novel RNAi reagent, targeting luciferase mRNA and human RPN2 mRNA (Supplementary Table 1, 2) were purchased from Bonac (Kurume, Japan). Allstars Negative Control siRNA was obtained from Qiagen (Hilden, Germany).

Preparation of novel RNAi agents

The preparation of proline diamide amidite and the novel RNAi agents has been previously described9. Novel RNAi agents are prepared as single-stranded RNA that self-anneals into a unique structure containing a double-stranded RNA with an unpaired site bound at the right and left ends by an oligonucleotide loop or by a non-nucleotide molecule (a proline derivative). Fig. 1 shows the schematic model of human RPN2, which was selected as a representative RNAi target.

RNA stability

To estimate the resistance to nucleases, 40 μl (2 μmol/l) of siRNA, nkRNA and PnkRNA directed against human RPN2 were incubated at 37°C with 1 μl of RNase Cocktail Enzyme Mix (Ambion, Foster City, CA) (RNase A 500 U/ml, RNase T1 20,000 U/ml). After the specified times, the ribonuclease reaction was stopped and 2 μl of each sample was run on 3% agarose gel.

Cell line

A549-luc-C8 cells, a luciferase-expressing cell line derived from A549 human lung adenocarcinoma cells by stable transfection of the North American Firefly Luciferase gene expressed from the CMV promoter, were purchased from Xenogen. The human lung adenocarcinoma cell line PC14 was obtained from RIKEN BioResource Center (Tokyo, Japan). These cells were cultured in RPMI-1640 containing 10% heat-inactivated FBS and an antibiotic-antimycotic at 37°C in 5% CO 2 .

RNA extraction

Total RNA was extracted from cultured cells or mice lungs using QIAzol and miRNeasy Mini Kit (Qiagen) according to the manufacturer's protocol. The purity and concentration of all RNA samples were quantified using NanoDrop ND-1000 (Thermo Scientific, San Jose, CA). For RPN2 mRNA analysis of mice lungs, the animals were sacrificed 24 h after the inhaled administration of each RNAi agent.

Quantitative Real-time PCR (qRT-PCR)

The reverse transcription reaction was performed with a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA) using a random hexamer primer. The synthesised cDNAs were quantified by SYBR Green I qRT-PCR. Quantitative real-time reverse transcription-PCR (qRT-PCR) analysis was conducted using primers for human RPN2 (forward:5′-CTTCCAGAGCCACTGTCCTC-3′; reverse: 5′-CCGGTTGTCACCTTCAACTT-3′). β-Actin (forward:5′-ATTGCCGACAGGATGCAGA-3′; reverse:5′-GAGTACTTGCGCTCAGGAGGA-3′) was used for normalisation. The relative amounts of RPN2 were measured using the 2(-Delta Delta C(T)) method. The reactions were performed with the ABI Prism 7300 Sequence Detection System (Applied Biosystems) at 95°C/10 min, followed by 40 cycles at 95°C/15 s and 60°C/30 s. All qRT-PCR reactions were performed in triplicate.

Transient transfection assays

A549-luc-C8 cells were plated on six-well plates at a density of 2 × 105 cells/well and grown overnight until 50–80% confluence was achieved to obtain maximum transfection efficiency. The cells were transfected with validated siRNA, the novel RNAi agents (PnkRNA, nkRNA) for RPN2, or Allstars Negative Control siRNA (Qiagen) at a final concentration of 25 nmol/l using the DharmaFECT 1 reagent (Thermo Scientific), according to the manufacturer's protocol. In the cDNA rescue experiment, an RPN2 Human cDNA ORF clone (Origene Technologies, Rockville, MD) or pEGFP-N1 (Clontech Laboratories, Mountain View, CA) was combined with each siRNA using DharmaFECT Duo (Thermo Scientific), according to the manufacturer's protocol. RPN2-siRNA targeting site is located 906 nt downstream of the ATG start codon of human RPN2 cDNA sequence on human RPN2 expression vector.

Cell Proliferation Assay (MTS assay)

A Cell Counting Kit-8 (CCK-8) (Dojindo Laboratories, Kumamoto, Japan) was used in the cell proliferation assay. Five thousand cells per well were seeded in 96-well plates. The following day, the cells were replenished with fresh medium containing 25 nmol/l of each RNAi agent. After four days of culture, a plate was assayed by adding 10 μl of CCK-8 solution to each well and the plate was further incubated for 4 h at 37°C. The absorbance at 450 nm was measured using Envision (PerkinElmer, Norwalk, CT).

RNAi therapeutic agent labelling with pHrodo™ Red succinimidyl ester and endocytosis assay

The fluorescence of the pHrodo™ Red dye increases as the pH decreases from neutral to acidic, making it an ideal tool to study endocytosis25. The amine-reactive forms of pHrodo™ Red succinimidyl ester were used for labelling the RNAi therapeutic agents. After being labeled according to the manufacturer's protocol, the oligonucleotides were purified by reverse-phase HPLC. A549-luc-C8 cells or PC14 cells were transfected with the labeled siRNAs or PnkRNAs. Each labeled oligonucleotide was transfected with or without DharmaFECT 1 reagent. Then, the plates were incubated at 37°C for 3 hours to allow endocytosis to run to completion. Hoechst 33342 was used as a DNA counter-stain (cyan). Microscopic analysis was performed with a FLUOVIEW FV10i confocal microscope.

In vivo imaging of RNAi therapeutic agents in mice with lung cancer

Animal experiments were performed in compliance with the guidelines of the Institute for Laboratory Animal Research, National Cancer Center Research Institute. These studies were approved by the National Cancer Center Research Institute. Six- to seven-week-old male C.B-17/Icr-scid/scidJcl mice (CLEA Japan, Shizuoka, Japan) were used in the experiments. The animals were housed in a 12 h light/12 h dark cycle and provided with an autoclaved rodent diet and water ad libitum. The mice were injected intravenously with 2 × 106 A549-luc-C8 cells suspended in 0.25 ml of sterile Dulbecco's PBS via the tail vein (day 0). For in vivo imaging, the mice were administered 150 mg/kg D-luciferin (Promega, Madison, WI) by intraperitoneal injection. Ten minutes later, photons from the whole bodies of the animals were counted by measuring bioluminescence with an IVIS imaging system (Xenogen, Alameda, CA), according to the manufacturer's instructions. The data were analysed using LIVINGIMAGE 4.2 software (Xenogen). The development of lung cancer was monitored twice a week in vivo by bioluminescent imaging. Four weeks after tumor injection (day 28), the bioluminescence from the implanted cancer cells was measured and the mice were divided into four treatment groups (single administration study, mice per group; n = 4) or two groups (repeated administration study, mice per group; n = 12) with equivalent levels of bioluminescence. In the single administration study, the individual mice were administered 15 μg of each RNAi agent with In vivo-jetPEI™ (Polyplus Transfection Inc, New York, NY) (resulting in a calculated 1:6 charge ratio of nucleic acid backbone phosphates to cationic lipid nitrogen atoms) in a volume of 25 μl on day 28 using an endotracheally inserted MicroSprayer™ aerosoliser (IA-1C; Penn-Century) and a high-pressure syringe (FMJ-250; Penn-Century, Philadelphia, PA)26. Data are from a representative experiment of three independent experiments. In the repeated administration study, the treatment—15 μg of each naked RNAi agent inhaled by using a MicroSprayer™ aerosoliser—was performed on days 28, 35 and 42 (once a week for 3 weeks, three treatments total). To control for mouse-to-mouse variability, the bioluminescence ratio for each mouse was normalised by dividing by each day of the post-treatment/pre-treatment (day28) ratio of luciferase intensity for that mouse. For in vivo knockdown analysis, animals were sacrificed 24 h after each RNAi application and lungs were removed and processed for histology or knockdown determination by qRT-PCR (SYBR Green).

Lung histological findings

Lung tissues were fixed in 10% neutral buffered formalin, paraffin-processed and sectioned at 5 μm. Formalin-fixed and paraffin-embedded slides were stained with haematoxylin and eosin (H&E) or used for immunohistochemical (IHC) staining. With regard to the histologic estimation of tumor burden, the freeware Image J (National Institutes of Health, Bethesda, Maryland, USA) was used. Upon the IHC staining, antigen retrieval was performed by autoclave in a 10 mmol/l sodium citrate buffer (pH 6.0) and the endogenous peroxidase activity was blocked with the Immuno Pure Peroxidase Suppressor (Pierce, Chester, UK). The slides were incubated with RPN2 (A-1, Santa Cruz Biotechnology, Santa Cruz, CA) or Ki-67 (M7240, Dako Cytomation, Copenhagen, Denmark) primary antibody at 4°C overnight. The next day, after washing, the samples were incubated with mouse peroxidase-conjugated anti-mouse IgG (ImmPRESS Reagent; Vector Labs, Burlingame, CA) for 1 h. The immunoreactions were visualised with diaminobenzidine and the sections were counterstained with haematoxylin.

Immunofluorescence staining

To estimate the inhaled distribution of RNAi agents, 15 μg of Allstars NegativesiRNA Alexia Fluor 647 (Qiagen) with In vivo-jetPEI™ was aerosolised by means of a MicroSprayer™ and the lungs were harvested 6 h after application, processed for paraffin sectioning and analysed by confocal microscopy. DAPI staining was carried out immediately before imaging. Imaging for the cyan (DAPI) and magenta (fluorescently labeled siRNA) channels was performed in sequential mode using the appropriate excitation and emission settings. Microscopic analysis was performed with a FLUOVIEW FV10i confocal microscope (OLYMPUS, Tokyo, Japan).

Statistical analysis

All experiments were repeated at least three times and the results are expressed as the means ± SE. The statistical analyses were conducted using the Bonferroni multiple-comparison test. These analyses were performed with the Expert StatView analysis software (version 4; SAS Institute, Cary, NC). P < 0.05 was considered to be statistically significant.