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BRC-mediated RNAi targeting of USE1 inhibits tumor growth in vitro and in vivo
Journal Pre-proof BRC-mediated RNAi targeting of USE1 inhibits tumor growth in vitro and in vivo Hyejin Kim, Yeon Kyung Lee, Kyung Ho Han, Hyunsu Jeon, In-ho Jeong, Sang-Yeob Kim, Jong Bum Lee, Peter C.W. Lee PII:
S0142-9612(19)30729-X
DOI:
https://doi.org/10.1016/j.biomaterials.2019.119630
Reference:
JBMT 119630
To appear in:
Biomaterials
Received Date: 16 October 2019 Accepted Date: 12 November 2019
Please cite this article as: Kim H, Lee YK, Han KH, Jeon H, Jeong I-h, Kim S-Y, Lee JB, Lee PCW, BRC-mediated RNAi targeting of USE1 inhibits tumor growth in vitro and in vivo, Biomaterials (2019), doi: https://doi.org/10.1016/j.biomaterials.2019.119630. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.
BRC-mediated RNAi targeting of USE1 inhibits tumor growth in vitro and in vivo
Hyejin Kim1, a, Yeon Kyung Lee2, a, Kyung Ho Han 2, Hyunsu Jeon1, In-ho Jeong 2, SangYeob Kim3, Jong Bum Lee1* and Peter C. W. Lee2*
1
Department of Chemical Engineering, University of Seoul, 163 Seoulsiripdaero,
Dongdaemun-gu, Seoul, Korea 2
Department of Biomedical Sciences University of Ulsan College of Medicine, Asan Medical
Center, Seoul, Korea 3
Department of Convergence Medicine, University of Ulsan College of Medicine, Asan
Medical Center, Seoul, Korea
*Corresponding author: *J. B. Lee, PhD, Department of Chemical Engineering, University of Seoul, 163 Seoulsiripdaero, Dongdaemun-gu, Seoul 02504, Korea. E-mail: [email protected]
*Peter C. W. Lee, PhD, Department of Biomedical Sciences, University of Ulsan College of Medicine, ASAN Medical Center, Seoul 05505, Korea. E-mail: [email protected]
Abstract USE1 has been demonstrated to play crucial roles in the development and progression of human lung cancer. However, the antitumor efficacy of RNA interference (RNAi) targeting of USE1 has not yet been evaluated as a possible clinical application. We here synthesized USE1 targeting bubbled RNA-based cargo (BRC) composed of densely packed multimeric pre-siRNAs with specific Dicer cleavage sites to enable efficient siRNA release upon entry to target cells. The physical entanglement and continuous networking of RNAs via hybridization during enzymatic replication serve as a driving force for the self-assembly of BRCs. These molecules effectively suppressed the transcription of their target genes, leading to tumor growth suppression in vitro and in vivo. Moreover, their repeated intravenous administration efficiently inhibited the growth of A549 tumor xenografts. Based on these findings of a reduced cancer cell viability following a USE1 knockdown, we further explored cell cycle arrest and apoptosis pathways. The observed tumor cell growth suppression was found to be controlled by cell cycle arrest and apoptosis signals induced by the USE1 reduction. These results suggest that USE1 BRCs may have future clinical applications as an RNAi-based cancer therapy.
Keywords: USE1, bubbled RNA-based cargo, RNAi, gene therapy, cancer
1. Introduction Lung cancer is one of the most common malignant tumors and plays a significant role in the worldwide cancer mortality rate. In 2011, the global number of lung cancer diagnoses and related deaths was about 1.6 million and 1.3 million, respectively [1]. Surgery and chemotherapy have been the most widely utilized treatments for lung cancer but the current survival rates and quality of life remain inadequate for patients with this disease [2]. Hence, the identification of novel therapeutic targets is highly desirable for these tumors. USE1 (UBA6-specific E2 conjugating enzyme 1), also known as ubiquitin-conjugating enzyme E2Z (UBE2Z), is a member of the E2 enzyme family. USE1 comprises an E2 catalytic core domain that contains an active site cysteine residue that is required for the formation of a thioester bond with ubiquitin [3, 4]. Interestingly, for ubiquitin conjugation USE1 functions specifically with the UBA6 ubiquitin-activating (E1) enzyme rather than the conventional UBE1 E1 enzyme [3, 4]. In our recent study, we analyzed the protein levels in lung cancer patients and found that USE1 was elevated. Consequently, we observed that the overexpression of USE1 significantly increased proliferation, migration, and invasion in both cells and animals, whilst a decrease in the USE1 levels in lung cancer cells reversed these effects suggesting that this factor is a possible cancer therapeutic target [5]. The therapeutic efficacies of USE1 have not been verified with lung cancer-specific compounds or as a gene therapy. We postulated that RNA molecules could verify the potential value of USE1 as a target for specific gene therapy against lung cancer. The principal benefits of RNAi therapy are its specificity, potency, and versatility [6]. RNAi technology can be applied as a novel therapy to the treatment of genetic disease, infectious disease and cancer [7]. One of the most promising recent advances in the synthesis of RNA-based structures is rolling circle transcript (RCT)-mediated enzymatic self-assembly
[8]. RCT-based enzymatic self-assembly has now been applied to various RNA structures for RNAi-based therapies, such as RNAi microsponges, siRNA nanosheets, and tumor-targeting RNA nanovectors. RNAi is affected by the Dicer cleavage efficiency, which is dependent on material design and fabrication criteria [9]. The efficiency of Dicer cleavage of RNAi microsponges was found to be about 21% but a recently developed siRNA nanosheet structure has improved Dicer-mediated functional siRNA release by more than 80% [9-11]. More recently, we described our new complementary rolling circle transcription (cRCT) technology to derive the self-assembly of bubbled RNA-based cargoes (BRCs) composed of specific Dicer cleaving sites for efficient siRNA release [9]. Moreover, the ability to control reaction conditions has enabled the synthesis of BRCs of optimal size for efficient cellular uptake [9, 12]. In our current study, BRCs were synthesized to target USE1 molecules. Their antitumor efficacies against various cancer cells, including lung cancer cells, were then assessed. Consistent with earlier in vitro results, we here confirm that BRC-mediated USE1 reduction causes apoptosis in lung tumor models bearing A549 cells. We conclude that USE1 silencing plays a leading role in lung tumor growth.
2. Materials and methods 2.1. Preparation and transfection of BRC molecules BRC molecules that silence USE1 (GenBank accession no. NM_153451) were used in the present study. A non-targeting BRC molecule was used as the negative control. For the fabrication of BRCs, linear and primer DNA (Integrated DNA Technologies) were hybridized and circularized following a previously described protocol [3]. Final sense- and antisensecircular DNA concentrations of 0.5 µM, 1 mM of each rNTP (New England BioLabs) and T7 RNA polymerase (80 U µl-1; AM2085; Invitrogen) were mixed with polymerase reaction buffer (40 mM Tris-HCl, 6 mM MgCl2, 1 mM DTT, 2 mM spermidine (pH 7.9 @ 25°C;
M0251S, New England BioLabs), and RCT reactions were carried out for 20 h at 37°C. For the synthesis of cy3-labeled BRCs, 50 µM of cy3-UTP (ENZ-42506; Enzo Life Sciences) was added to the reaction. For the synthesis of SYBR Green II-labeled BRCs, SYBR Green II 10,000X (Invitrogen) was mixed with the BRCs at a final concentration of 10X and incubated at room temperature for 10 min. To remove excess unreacted monomers or dye molecules, the final reaction solution was briefly sonicated and the BRCs were washed three times with nuclease-free water prior to further analysis. For BRC transfection, RNAimax (Invitrogen) was used following the manufacturer’s protocol.
2.2. Characterization of BRCs FE-SEM (Hitachi) was employed to observe the surface morphology of the synthesized BRC molecules. For SEM imaging, the samples were deposited onto a silicon wafer, air-dried, and coated with Pt. AFM (Park Systems) was used to obtain high z-resolution images of the BRCs. The BRCs used for AFM imaging were deposited onto freshly cleaved mica and airdried. The samples were then scanned in non-contact mode with a PPP-NCHR tip (Park Systems) and the AFM image was processed with XEI software (Park Systems). Dynamic light scattering with a ZetaSizer ZS90 (Malvern Panalytical) was employed to analyze size distribution and polydispersity index for the BRCs before and after complexation with RNAimax reagent. Nanoparticle tracking analysis (NTA) was carried out with NanoSight NS300 (Malvern Panalytical) with a 565 nm filter and the data were analyzed with NanoSight software.
2.3. Stability of BRCs To assess the stability of BRCs, the nanoparticles and ssRNA mixture were incubated at 37°C with 10% fetal bovine serum-containing media (FBS; Gibco) for 30 min, 6 h, 12 h and
24 h. The RNA samples, including untreated BRCs, were resolved in 10% non-denaturing polyacrylamide gel electrophoresis (PAGE) gels at 85 V for 70 min in 1X TBE buffer (BioRad). The gels were then stained with 1 X GelRed (41003, Koma Biotech) in 1X TBE and analyzed under UV with a GelDoc EZ Imager (Bio-Rad). This experiment was carried out three times and the original images of the gels are shown in Figure 1F and Supplemental Figure 2. The band intensities were analyzed using the GelDoc software and plotted as shown in Figure 1G.
2.4. Cell lines and cell culture All cells were obtained from ATCC and were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS and maintained in a 5% CO2 incubator at 37°C.
2.5. Western blotting For western blotting analysis, tissues and cells were homogenized in lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, 0.5% Triton X-100, 1 mM EDTA) supplemented with protease inhibitors (Roche). The lysates were then centrifuged at 13,500 g for 30 min and the supernatants were recovered. About 30 µg of protein extracts were resolved by SDS-PAGE and analyzed by western blotting using primary antibodies against UBA6 [4], USE1 [5], Cyclin D (2978, Cell Signaling Technology), Cyclin E (4129, Cell Signaling Technology), Caspse-3 (9662, Cell Signaling Technology), p53 (sc-126, Santa Cruz Biotechnology), Bax (sc-7480, Santa Cruz Biotechnology), α-β-actin (sc-47778, Santa Cruz Biotechnology), and α-alpha-tubulin (ab7291, Abcam). This was followed by incubation with a corresponding IgG horseradish peroxidase-conjugated secondary antibody (ThermoFisher Scientific) and detection using enhanced chemiluminescence (Bio-Rad).
2.6. Apoptosis and cell cycle analysis Apoptosis analysis was carried out using an annexin V–FITC Apoptosis Detection Kit (Invitrogen). Briefly, cells (4 × 105) were exposed to 2 µg of scrambled control and USE1 BRC molecules for 48 h. The cells were then collected by centrifugation and resuspended in 500 µl of 1x binding buffer, followed by the addition of annexin V–FITC (5 µl) and PI (5 µl). After a short incubation at room temperature for 5 min in the dark, the cells were analyzed by FACS using a flow cytometer (FACSCalibur; BD Biosciences). The cells that stained positively for early apoptosis (annexin V–FITC-stained only) and for late apoptosis (annexin V–FITC- and PI-stained) were combined for analysis. Propidium iodide (PI) staining was used to analyze the DNA content and cell cycle distribution. After exposure to different concentrations of magnolol for 24 h, the cells were harvested and fixed in 70% ethanol, centrifuged (1500 rpm, 5 min), incubated with RNase (100 mg⁄mL) at 37°C for 30 min, and stained with PI (50 mg⁄mL in PBS). The cellular DNA content and cell cycle distribution were then analyzed by flow cytometry (FACSCalibur).
2.7. Cell proliferation assay For siRNA transient transfection assays, cells were seeded in 10 cm dishes at a density of 5 × 106 cells/dish and transfected with 20 nM siCDH17 or scrambled siRNA. After a 48 h incubation, the cells were disassociated and transferred to a 96-well plate at a density of 3000 cells/well, and then incubated for another 72 h. Cell viability was assayed using a Cell Counting Kit (CCK)-8 (Donjindo). For BRC transfection assays, the cells were seeded into 6 well plates at a density of 4 × 105cells/well and transfected with 2 µg USE 1 BRC or the scrambled BRC control. After 48 h of incubation, the cells were disassociated and transferred to 96-well plates at a density of 1000 cells/well, then incubated for another 0, 24, or 48 h.
Cell viability was assayed again using the CCK-8 kit. The absorbance (λ, 450 nm) of the resulting solution was measured with a spectrophotometer.
2.8. Scratch wound-healing assay For a scratch wound-healing assay, the cells were first seeded at 5 × 105 cells per well in 6well plates and incubated. Cell monolayers were then scratched with a sterile 200 µl pipette tip and washed with medium to remove detached cells. Cell migration was evaluated by measuring the differences in the wound areas on the monolayer.
2.9. Invasion assay To measure invasiveness, the cells were seeded at 1 × 105 cells per well in the upper chamber of a transwell insert (8-µm polycarbonate membrane; Corning) coated with 1–2 mg of matrigel (BD Biosciences) that was placed into a 24-well plate. The lower chamber was then filled with 800 µl DMEM supplemented with 10% fetal bovine serum and incubated for 36 h. Cells that invaded the matrigel to the underside of the membrane were fixed with 4% paraformaldehyde and stained with 0.5% crystal violet for 20 min. Cells that remained at the upper surface of the insert were removed with a cotton swab. The invading cells were counted in five random fields under a light microscope.
2.10. Colony formation assay To assess colony formation, cells were seeded at 1 × 103 cells per well in 6-well dishes and cultured in DMEM supplemented with FBS for 2 weeks. The cells were then fixed with 4% paraformaldehyde and stained with 0.5% crystal violet for 30 min.
2.11. Animals and tumor model
Female BALB/c nude mice (5 weeks old) were purchased from Orient Bio Inc. All animal studies were performed in accordance with the Institutional Ethics Committee and Institutional Animal Care Committee of University of Ulsan College of Medicine (2017-12281). A549 cells (1 × 107) were injected subcutaneously into the left flank of the mice. Tumor volumes (V) were then measured by V = L (w)2 × 0.5, where L is the length and w is the width of tumor.
2.12. In vivo therapeutic efficacy The antitumor effects of the USE1 BRC particles were investigated in lung cancer xenografts comprising A549 cells. When the tumor volumes reached a size of about 100 mm3, the mice were randomly split into 3 groups (n=5 per group). The animals were then anesthetized and administered intravenously with PBS, a scrambled BRC control, or USE1 BRC, respectively. The concentrations of the scrambled and USE1 BRC molecules were normalized to 5 µg in 0.1 mL PBS solution. BRC particles were injected intravenously three times in a week for 3 weeks. Tumor growth was recorded for 17 days. To further analyze any antitumor effects, the tumors were excised at 17 days after treatment and stained with hematoxylin and eosin (H&E). To visualize USE1 expression in the tumor tissues, immunohistochemistry with a USE1-specific antibody was conducted (final dilution 1:200). The slides were incubated overnight at 4°C and immunostaining was performed using a commercially available kit (Vectastain Universal Quick Kits and DAB, vector lab). A terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was carried out using a TdT-FragEL DNA fragmentation detection kit in accordance with the manufacturer’s instructions (Merck) to detect apoptotic cell death areas.
2.13. Biodistribution
Mice were injected via the tail vein with 100 µl of 15 µg SC BRC-Cy3, USE1 BRC-Cy3 or PBS. At 30 mins or 18 hours after injection (n= 3), the animals were euthanized and tissues were harvested including the heart, liver, lung, kidney and spleen. Each organ was imaged using the IVIS system (Perkin-Elmer). The average fluorescence intensity was determined for each tissue type using the same threshold settings after normalization with control organs.
2.14. Toxicity of the BRC molecules To assess the toxicity of the BRCs in vivo, additional mice were injected with 25ug SC BRC, USE1 BRC or PBS via the tail vein. At 24 hours after injection (n= 5), these animals were euthanized and the lung, liver, spleen and kidney were harvested and fixed in 10% formalin. The tissues were then paraffin embedded and H&E stained according to standard protocols. Each tissue was analyzed by a veterinary pathologist.
2.15. Biochemical assays Mouse blood samples were collected 24 hours after tail vein injection and then centrifuged. The serum obtained was stored at −20°C until analysis. The serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatinine (CREA), blood urea nitrogen (BUN), total protein (TP), and total bilirubin (TBIL) were then assayed using a Hitachi clinical analyzer 7180 (Hitachi). A complete blood count (CBC) test was performed using an Advia 2120i automated analyzer (Siemens).
3. Results 3.1. BRCs potentiate RNAi through their carefully engineered design By utilizing the recent advances in RNA nanotechnology, we fabricated BRCs using a complementary rolling circle transcription (cRCT) reaction to enable the efficient down-
regulation of USE1 expression [9, 13]. Based on previous findings, BRCs have doublestranded regions regularly spaced by a bubble region which enables the efficient release of siRNAs through cleavage by Dicer (Fig. 1A). The particle size was successfully engineered down to 350 nm in our previous study by manipulating the enzyme to template DNA ratios, but we aimed to further reduce this size in our present study. The self-assembly of the transcripts into 200 nm-sized particles was achieved by limiting the concentration of monomers in the cRCT reaction, whilst keeping the same 160:1 ratio of replicating enzyme to template DNA.
3.2. RNAs are packaged into BRCs with a highly porous structure and an adequate size for RNA delivery into tumor cells The highly porous surface morphology of our self-assembled BRC structure was revealed by scanning electron microscopy (SEM) (Fig. 1B). The high surface area of this structure facilitates easier access by the siRNA-releasing enzyme upon internalization into the target cells. Our average overall BRC size of 200 nm was further confirmed by atomic force microscopy (AFM; Fig. 1C), and the polydispersity index of these molecules (PDI; 0.150) measured by dynamic light scattering (DLS) indicated their monodispersity (Fig. 1D). It should also be noted that the sizes of these BRCs were within the favorable range for tumor site infiltration [14]. The increase in the hydrodynamic size indicated successful lipoplex formation upon introducing the transfection reagent, with the PDI remaining near 0.2, suggesting homogenous complexes were formed stably without aggregation (Supplemental Fig. 1). To confirm that the cRCT-induced nanostructure was generated from the RNA strands, a fluorescently labeled nucleotide triphosphate (cyanine 3-labeled UTP; cy3-UTP) was introduced to the cRCT reaction. The replicated RNA strands were then cy3 labeled by
incorporation of the cy3-UTP nucleotides by T7 RNA polymerase activity during the cRCT reaction. These fluorescently tagged nanostructures were evaluated by nanoparticle tracking analysis (NTA) which enables the measurement of nanoparticle size distribution and concentration. There was no significant difference found in the concentrations of the cy3labeled BRCs measured with the fluorescence and non-fluorescence mode of NTA, indicating that these molecules had been generated from cRCT reaction transcripts (Fig. 1E). Whilst the small size and porous structure of our BRC molecules facilitated a more optimal internalization into cancer cells and the ready access of the Dicer enzyme to release functional siRNAs, their compact structure also provided high physiological stability. This stability was assessed in the presence of 10% fetal bovine serum for different time periods i.e. from 30 min to 24 h (Fig. 1F, Supplemental Fig. 2). The results indicated that over 50% of the BRCs retained their molecular weights after 24 h of serum treatment, in contrast to the majority of an ssRNA mixture which was degraded within 30 min in the presence of serum (Fig. 1G).
3.3. A USE1 knockdown in both HeLa and A549 cells reduces cell proliferation and colony formation Since deregulated cell proliferation and enhanced colony formation are well established features of cancer [15-17], we tested the impact of USE1 silencing on A549 and HeLa cell growth in a CCK-8 assay. The results indicated in the first instance that USE1 BRCs were effective in suppressing the growth of these different cancer cell lines at the protein level (Fig. 2A, and Supplemental Fig. 3). The proliferative activities of the A549 and HeLa cells were found to gradually decrease when treated with USE1 BRC compared to the PBS and scrambled BRC controls (Fig. 2B, and Supplemental Fig. 4). We next evaluated the colony formation capacity of these cells under the same treatment conditions. As shown in Figure 2C,
the USE1 BRC-treated groups for both cell types had the lowest colony formation.
3.4. USE1 regulates the migration and invasion of HeLa and A549 cells A previous study has reported that USE1 is significantly associated with migration and invasiveness in lung cancer [5]. A scratch wound healing assay was performed to assess the migration-promoting ability of USE1 in A549 and HeLa cells, again using BRC particles to silence the gene (Fig. 3A and B). Both cell lines transfected with USE1 BRC molecules showed significantly reduced cell migration in a time-dependent manner, whereas no reduction was seen in the scrambled BRC control groups. In a matrigel invasion assay, the USE1 BRC-treated cells of both lines exhibited impaired invasiveness compared with the control groups (Fig. 3C). Taken together, these results indicated that the inhibition of USE1 impedes the migration and invasiveness of cancer cells.
3.5. USE1 silencing induces apoptosis and S phase cell cycle arrest in HeLa and A549 cells To confirm whether the proliferation suppression of HeLa and A549 cells caused by the RNAi silencing of USE1 was due to an apoptotic response, we analyzed these cells by flow cytometry using annexin V and PI staining. The apoptosis rate (annexin-V +/PI - and annexin-V+/PI +) of both USE1 BRC-treated cell lines at 48 h post transfection was indeed found to be elevated over the controls. The inhibition of USE1 significantly increased the apoptotic percentage in the HeLa and A549 cell populations by 48.8% and 37%, respectively, compared with the blank and scrambled BRC control cells (Fig. 4A). To further investigate the potential mechanisms by which USE1 silencing controls cancer cell growth, we examined the cell cycle of both the HeLa and A549 cell lines by flow cytometry following transfection with USE1 BRC particles. At 48 h post-transfection, the
proportion of USE1 BRC-treated HeLa cells in G1- and S-phase was reduced compared to the blank and scrambled controls. The percentage of cells in the sub G1-phase for the control, scrambled BRC-treated and USE1 BRC-treated HeLa cells was 0.3%, 2.8%, and 17.3%, respectively (Fig. 4B). The percentage of scrambled BRC-transfected A549 cells in S-phase was about 15-29%, whereas this was about 40% of the USE1 BRC-transfected A549 cells. At the same time, the number of USE1 BRC-treated A549 cells in the sub-G1 phase was higher than the two control groups (Fig. 4B). In addition, a downregulation of both cyclin D and E was detected (Fig. 4C). The protein levels of Bax and cleaved cas-3 were also found to be activated upon USE1 inhibition. Taken together, these results suggest that USE1 silencing suppresses the proliferation of cancer cells in vitro through cell cycle arrest and the induction of apoptosis.
3.6. Antitumor efficacy of USE1 BRC particles in an A549 tumor xenograft model The therapeutic effects of our USE1-targeting BRC particles were further examined in vivo using mice harboring A549 lung tumor xenografts. USE1 BRC particles, PBS, or a scrambled BRC control were injected intravenously into these tumors when the volume reached approximately 100 mm3. A total BRC amount of 0.025 mg/kg was administered three times a week for 3 weeks into the animals. Tumor growth was measured for 17 days after these treatments and the USE1 BRC injection resulted in a smaller tumor size compared with the controls (Fig. 5A). As shown in Figure 5B, the scrambled BRC control mice tumor volumes were comparable to those in the PBS control group. At day 10 post-treatment, the USE1targeted BRC group showed about a 70% tumor growth suppression compared with the two control groups (P < 0.05). To investigate the possible mechanisms of tumor growth inhibition in these experiments, USE1, p53, Bax and cleaved cas-3 were evaluated in the lung tumors by western blotting (Fig.
5D). The cellular concentrations of the USE1 proteins in the same tumor tissues were confirmed as being efficiently reduced by the administration of USE1 BRC. However, the expression levels of p53, Bax and cleaved cas-3, which are known signaling proteins for apoptotic cell death, were notably increased. The non-targeting scrambled BRC particles did not produce these effects under the same transfection conditions. To evaluate the antitumor activity of each treatment, histological examinations of the tumor tissue sections were performed using hematoxylin and eosin (H&E) staining and a TUNEL assay (Fig. 5E). The results revealed that cell death lesions and apoptotic tumor cells were localized in the tumors treated with USE1 BRC or scrambled BRC. However, the tumors in the mice treated with USE1 BRC showed increased cell death and larger apoptotic areas compared to the scrambled BRC control group. Furthermore, a reduced number of USE1 areas were detected in the USE1 BRC-treated group (Fig. 5E).
3.7. Biodistribution and toxicity of USE1 BRC molecules To investigate the biodistribution and toxicity of the USE1-targeting BRC particles in mice, USE1 BRC-Cy3 particles, PBS, or a scrambled BRC-Cy3 control were injected via the tail vein. After administration, BRC particle accumulation in different organs was detected using the IVIS system. A significant accumulation was observed in the kidney of USE1 BRC- and scrambled BRC-injected mice at 30 mins after injection (Fig. 6A). Interestingly, only USE1 BRC-Cy3 nanoparticles were observed in the kidney up to 18 hours after injection (Supplemental Fig. 5A). To assess the potential toxic effects of USE1 BRC molecules in the mice, blood biochemistry tests were conducted but showed that no significant differences between the groups (Fig. 6B). Subsequent histological analysis showed that the USE1 BRCs did not cause significant injury to the liver, kidney, spleen or lung tissues (Fig. 6B). These findings
indicated no induction of toxicity by USE1 BRC in vivo.
4. Discussion Our previous study implicated USE1 in lung cancer development and we reported that its overexpression promoted the proliferation, migration, and invasion of lung tumor cells [5]. USE1 was thus considered to be a molecular regulator of tumor growth in lung cancer. It is well known that most cancer types are defined by augmented cell proliferation and a reduced apoptotic response. Identifying molecular regulators of tumor cell growth is therefore an important part of cancer research. We have here presented the potency of BRCs in silencing USE1 via RNAi and assessed the effects of this treatment in the HeLa cervical cancer and A549 lung cancer cell lines to assess its antitumor efficacy. Our present results clearly indicate that the knockdown of USE1 using targeted BRCs significantly suppresses tumor cell growth both in vitro and in vivo. Mechanistically, we demonstrated an induction of S-phase cell cycle arrest and an apoptotic response through the p53 signaling pathway in cells transfected with USE1 BRCs. BRC control molecules had no toxic effects in the cells. Our findings therefore indicate that these molecules have potential clinical application as a new form of cancer therapy. Apoptosis is a well-known process for eliminating unhealthy cells [18] and is triggered by two distinct signaling pathways [19-22]. One of the major apoptotic pathways in cancer cells occurs in mitochondria [23] and we observed this process in A549 and HeLa cells after transfection of USE1 BRCs, with the consequent decrease in cell proliferation. In further functional studies, we assayed the levels of p53 and Bax, a pro-apoptotic factor [24], and also caspase-3, an apoptotic effector protein [25-29]. The USE1 BRC knockdown significantly augmented p53 and Bax expression. The caspases are another important activating component of p53-mediated apoptosis [30] and caspase-3 was found in our experiments to be
expressed at higher levels following treatment with USE1 BRCs. Cell cycle arrest is another pathway that can lead to apoptosis and is commonly deregulated in cancer [31, 32]. Consistently, the USE1 BRCs caused an S-phase arrest in both A549 and HeLa cells. In support of these findings, the critical regulator of S-phase arrest, cyclin E [33-35], along with the other crucial cell cycle factor cyclin D1, were found to be clearly suppressed by USE1 BRC transfection, indicating that the downstream effectors of USE1 are these cyclins. We employed an A549 xenograft model system to investigate whether the effects of the USE1 BRCs in vitro would be reproduced in vivo. The mice treated with USE1 BRCs showed a greater reduction of tumor growth than either the scrambled BRC or PBS control groups. Moreover, H&E staining and TUNEL assays of the USE1-suppressed A549 tumor sites revealed regions of necrosis and apoptosis. Of note, a lower expression of USE1 was confirmed in the USE1 BRC group. These findings were consistent with the cell cycle arrest and apoptosis caused by USE1 silencing in vitro. Moreover, when we further analyzed the key apoptotic factors p53, Bax, and caspase-3, we observed higher expression levels in tumor tissues in which USE1 had been silenced. In conclusion, USE1 targeting by BRCs in both cervical and lung cancer cells in vitro, and in A549 lung cancer xenografts in vivo, shows promise as an anti-cancer therapeutic strategy. Mechanistically, the suppression of cancer cell proliferation resulting from the USE1 knockdown was caused by a cell cycle arrest in S-phase and an enhanced apoptotic response. We propose that BRC-based therapies may prove to be effective in the future against various solid cancers.
AUTHOR INFORMATION Corresponding authors email: [email protected] and [email protected]
Conflict of interest declaration The authors declare no competing financial or other interests in relation to this article.
Acknowledgements This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (NRF-2019R1A2C2084181), and also supported by the Bio & Medical Technology Development Program of the National Research Foundation (NRF) funded by the Ministry of Science & ICT (2016M3A9C6917402).
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Table 1. DNA sequences used to generate USE1-targeting BRCs. /5Phos/ indicates 5’ phosphorylation. The primer binding, USE1 pre-siRNA, and non-targeting pre-siRNA regions are indicated in purple, red, and orange, respectively.
Name USE1 siRNA targeting region in the mRNA Primer for the T7 promoter region
Sequence (5’ → 3’) (mRNA) GGG AAA GUC UGC UUG AGU A TAA TAC GAC TCA CTA TAG GGA T
USE1 pre-siRNA sense strand
/5Phos/ ATA GTG AGT CGT ATT AAA GGG AAA GTC TGC TTG AGT ATT GCT GGA TGA AGG ACG GTC GAA CGC AAG GGA AAG TCT GCT TGA GTA TTA TCC CT
USE1 pre-siRNA antisense strand
/5Phos/ ATA GTG AGT CGT ATT AAA TAC TCA AGC AGA CTT TCC CTT CTT AGG CTG GAC AAC AAC CAT CTA AAT ACT CAA GCA GAC TTT CCC TTA TCC CT
Non-targeting presiRNA sense strand
/5Phos/ ATA GTG AGT CGT ATT AAA GTA TCT CTT CAT AGC CTT ATT GTA TCT CTT CAT AGC CTT AAA GTA TCT CTT CAT AGC CTT ATT ATC CCT
Non-targeting presiRNA antisense strand
/5Phos/ ATA GTG AGT CGT ATT AAA TAA GGC TAT GAA GAG ATA CTT GTA TCT CTT CAT AGC CTT AAA TAA GGC TAT GAA GAG ATA CTT ATC CCT
Figure Legends
Fig. 1. (A) Schematic illustration of the synthesis of USE1-targeting BRCs. (B) Low and high magnification (inset) SEM images of the BRCs, revealing their spherical shape and porous surface. (C) 3D reconstructed AFM image of the BRCs suggesting that their overall zdimensional sizes are ~200 nm. (D) Size distribution and polydispersity index of the BRCs analyzed by DLS, indicating their monodispersity. (E) Concentrations of cy3-labeled BRCs measured by NTA with or without a 565 nm filter (student’s t test; n.s., not significant). (F) Stability analysis of ssRNA and BRCs in the presence of 10% FBS, analyzed by nondenaturing PAGE. (G) Band intensity analysis of the polyacrylamide gels in panel (F) and in Supplemental Figure 2.
Fig. 2. A USE1 knockdown reduces the proliferation and colony formation capacity of HeLa and A549 cells. (A) USE1 protein levels in HeLa and A549 cells upon transfection with 2 µg USE1 BRC for 48 h, analyzed by western blot. Scrambled BRC transfected cancer cells (2 µg) and blank cells were used as controls. (B) Effects of USE1 silencing on HeLa and A549 proliferation, detected by CCK-8 assay. Results represent the means ± SE (n = 4); ***P < 0.001. (C) Effects of USE1 silencing on the colony formation ability of HeLa and A549 cells, tested using a plate colony assay. The relative colony formation efficiency of the blank cells was set as 1. Each column represents the mean value from triplicate experiments in each group. Data are the mean ± SE; *P < 0.001.
Fig. 3. USE1 regulates the migration and invasion of HeLa and A549 cells. (A, B) The migration ability of HeLa and A549 cells was assessed using an in vitro scratch wound healing assay. Images were taken 0, 24, and 48 h after wounding of the cell monolayers. (C, D) The invasiveness of the HeLa and A549 cells was evaluated using a Transwell assay, and images were captured at 48 h after incubation of the cells in a matrigel-precoated Transwell chamber (original magnification, × 200); ***P <0.001 compared with scrambled BRC-treated cancer cells.
Fig. 4. A USE1 knockdown in HeLa and A549 cells induces apoptosis and S-phase cell cycle arrest. (A) Apoptotic cells was detected by annexin V and PI double-staining. A total of 2 µg of USE1 BRC was transfected into the cells for 48 h. (B) The cell cycle distribution was assessed by flow cytometry at 48 h post-treatment. (C) Effect of USE1 silencing on the expression of cyclin D1, cyclin E, Bax, cleaved cas-3 and USE1. β-actin was used as an internal loading control.
Fig. 5. Antitumor efficacy of USE1 BRC particles in an A549 xenograft model. (A) Dissected tumors in each treatment group are shown at 17 days post-treatment. The A549 xenografts in the mice were injected intravenously with PBS, scrambled BRC or USE1 BRC three times per week for three weeks. (B) Tumor growth was measured over 17 days postinjection by assessing the tumor volumes using the formula: L (w)2 × 0.5, where L is the largest and w is the smallest diameter. Data represent the mean ± SE (n = 5); **P < 0.01, ***P < 0.001 versus the Control and Scrambled BRC groups. (C) Changes in the mouse body weights during the aforementioned treatments. (D) USE1 silencing is associated with the activation of p53, Bax and caspase-3 in the A549 xenograft tumors. (E) Histological analysis of xenograft tumor tissues treated with PBS, scrambled BRC, or USE1 BRC. Tumor tissues stained with H&E, a USE1-specific antibody, and TUNEL were viewed under a light microscope. Original magnifications in all three cases, × 100. Scale bar, 10 µm.
Fig. 6. Ex vivo optical images and biochemical toxicity of USE1 BRC in mice. (A) Biodistribution of USE1 BRC particles in the mice. The mice were injected intravenously with PBS, scrambled BRC-Cy3 or USE1 BRC-Cy3. The biodistribution of the BRCs in selected organs (liver, lung, heart, spleen and kidney) was analyzed using the IVIS system at 30 min after injection; n=3 in each group. (B) Effect of USE1 BRC particles injection in mice. H&E staining, complete blood count (CBC) and various biochemical parameters were evaluated, and data were expressed as a median ± standard deviation; n=5 in each group. The units followed by WBC (103/µL), RBC (106/ µL), HGB (g/dL), PLT (103/µL), AST (U/L), ALT (U/L), BUN (mg/dL), TP (g/dL), TBIL (mg/dL) and CREA (mg/dL). Original magnifications in all three cases, × 100. Scale bar, 10µm.
Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
S0142-9612(19)30729-X
DOI:
https://doi.org/10.1016/j.biomaterials.2019.119630
Reference:
JBMT 119630
To appear in:
Biomaterials
Received Date: 16 October 2019 Accepted Date: 12 November 2019
Please cite this article as: Kim H, Lee YK, Han KH, Jeon H, Jeong I-h, Kim S-Y, Lee JB, Lee PCW, BRC-mediated RNAi targeting of USE1 inhibits tumor growth in vitro and in vivo, Biomaterials (2019), doi: https://doi.org/10.1016/j.biomaterials.2019.119630. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.
BRC-mediated RNAi targeting of USE1 inhibits tumor growth in vitro and in vivo
Hyejin Kim1, a, Yeon Kyung Lee2, a, Kyung Ho Han 2, Hyunsu Jeon1, In-ho Jeong 2, SangYeob Kim3, Jong Bum Lee1* and Peter C. W. Lee2*
1
Department of Chemical Engineering, University of Seoul, 163 Seoulsiripdaero,
Dongdaemun-gu, Seoul, Korea 2
Department of Biomedical Sciences University of Ulsan College of Medicine, Asan Medical
Center, Seoul, Korea 3
Department of Convergence Medicine, University of Ulsan College of Medicine, Asan
Medical Center, Seoul, Korea
*Corresponding author: *J. B. Lee, PhD, Department of Chemical Engineering, University of Seoul, 163 Seoulsiripdaero, Dongdaemun-gu, Seoul 02504, Korea. E-mail: [email protected]
*Peter C. W. Lee, PhD, Department of Biomedical Sciences, University of Ulsan College of Medicine, ASAN Medical Center, Seoul 05505, Korea. E-mail: [email protected]
Abstract USE1 has been demonstrated to play crucial roles in the development and progression of human lung cancer. However, the antitumor efficacy of RNA interference (RNAi) targeting of USE1 has not yet been evaluated as a possible clinical application. We here synthesized USE1 targeting bubbled RNA-based cargo (BRC) composed of densely packed multimeric pre-siRNAs with specific Dicer cleavage sites to enable efficient siRNA release upon entry to target cells. The physical entanglement and continuous networking of RNAs via hybridization during enzymatic replication serve as a driving force for the self-assembly of BRCs. These molecules effectively suppressed the transcription of their target genes, leading to tumor growth suppression in vitro and in vivo. Moreover, their repeated intravenous administration efficiently inhibited the growth of A549 tumor xenografts. Based on these findings of a reduced cancer cell viability following a USE1 knockdown, we further explored cell cycle arrest and apoptosis pathways. The observed tumor cell growth suppression was found to be controlled by cell cycle arrest and apoptosis signals induced by the USE1 reduction. These results suggest that USE1 BRCs may have future clinical applications as an RNAi-based cancer therapy.
Keywords: USE1, bubbled RNA-based cargo, RNAi, gene therapy, cancer
1. Introduction Lung cancer is one of the most common malignant tumors and plays a significant role in the worldwide cancer mortality rate. In 2011, the global number of lung cancer diagnoses and related deaths was about 1.6 million and 1.3 million, respectively [1]. Surgery and chemotherapy have been the most widely utilized treatments for lung cancer but the current survival rates and quality of life remain inadequate for patients with this disease [2]. Hence, the identification of novel therapeutic targets is highly desirable for these tumors. USE1 (UBA6-specific E2 conjugating enzyme 1), also known as ubiquitin-conjugating enzyme E2Z (UBE2Z), is a member of the E2 enzyme family. USE1 comprises an E2 catalytic core domain that contains an active site cysteine residue that is required for the formation of a thioester bond with ubiquitin [3, 4]. Interestingly, for ubiquitin conjugation USE1 functions specifically with the UBA6 ubiquitin-activating (E1) enzyme rather than the conventional UBE1 E1 enzyme [3, 4]. In our recent study, we analyzed the protein levels in lung cancer patients and found that USE1 was elevated. Consequently, we observed that the overexpression of USE1 significantly increased proliferation, migration, and invasion in both cells and animals, whilst a decrease in the USE1 levels in lung cancer cells reversed these effects suggesting that this factor is a possible cancer therapeutic target [5]. The therapeutic efficacies of USE1 have not been verified with lung cancer-specific compounds or as a gene therapy. We postulated that RNA molecules could verify the potential value of USE1 as a target for specific gene therapy against lung cancer. The principal benefits of RNAi therapy are its specificity, potency, and versatility [6]. RNAi technology can be applied as a novel therapy to the treatment of genetic disease, infectious disease and cancer [7]. One of the most promising recent advances in the synthesis of RNA-based structures is rolling circle transcript (RCT)-mediated enzymatic self-assembly
[8]. RCT-based enzymatic self-assembly has now been applied to various RNA structures for RNAi-based therapies, such as RNAi microsponges, siRNA nanosheets, and tumor-targeting RNA nanovectors. RNAi is affected by the Dicer cleavage efficiency, which is dependent on material design and fabrication criteria [9]. The efficiency of Dicer cleavage of RNAi microsponges was found to be about 21% but a recently developed siRNA nanosheet structure has improved Dicer-mediated functional siRNA release by more than 80% [9-11]. More recently, we described our new complementary rolling circle transcription (cRCT) technology to derive the self-assembly of bubbled RNA-based cargoes (BRCs) composed of specific Dicer cleaving sites for efficient siRNA release [9]. Moreover, the ability to control reaction conditions has enabled the synthesis of BRCs of optimal size for efficient cellular uptake [9, 12]. In our current study, BRCs were synthesized to target USE1 molecules. Their antitumor efficacies against various cancer cells, including lung cancer cells, were then assessed. Consistent with earlier in vitro results, we here confirm that BRC-mediated USE1 reduction causes apoptosis in lung tumor models bearing A549 cells. We conclude that USE1 silencing plays a leading role in lung tumor growth.
2. Materials and methods 2.1. Preparation and transfection of BRC molecules BRC molecules that silence USE1 (GenBank accession no. NM_153451) were used in the present study. A non-targeting BRC molecule was used as the negative control. For the fabrication of BRCs, linear and primer DNA (Integrated DNA Technologies) were hybridized and circularized following a previously described protocol [3]. Final sense- and antisensecircular DNA concentrations of 0.5 µM, 1 mM of each rNTP (New England BioLabs) and T7 RNA polymerase (80 U µl-1; AM2085; Invitrogen) were mixed with polymerase reaction buffer (40 mM Tris-HCl, 6 mM MgCl2, 1 mM DTT, 2 mM spermidine (pH 7.9 @ 25°C;
M0251S, New England BioLabs), and RCT reactions were carried out for 20 h at 37°C. For the synthesis of cy3-labeled BRCs, 50 µM of cy3-UTP (ENZ-42506; Enzo Life Sciences) was added to the reaction. For the synthesis of SYBR Green II-labeled BRCs, SYBR Green II 10,000X (Invitrogen) was mixed with the BRCs at a final concentration of 10X and incubated at room temperature for 10 min. To remove excess unreacted monomers or dye molecules, the final reaction solution was briefly sonicated and the BRCs were washed three times with nuclease-free water prior to further analysis. For BRC transfection, RNAimax (Invitrogen) was used following the manufacturer’s protocol.
2.2. Characterization of BRCs FE-SEM (Hitachi) was employed to observe the surface morphology of the synthesized BRC molecules. For SEM imaging, the samples were deposited onto a silicon wafer, air-dried, and coated with Pt. AFM (Park Systems) was used to obtain high z-resolution images of the BRCs. The BRCs used for AFM imaging were deposited onto freshly cleaved mica and airdried. The samples were then scanned in non-contact mode with a PPP-NCHR tip (Park Systems) and the AFM image was processed with XEI software (Park Systems). Dynamic light scattering with a ZetaSizer ZS90 (Malvern Panalytical) was employed to analyze size distribution and polydispersity index for the BRCs before and after complexation with RNAimax reagent. Nanoparticle tracking analysis (NTA) was carried out with NanoSight NS300 (Malvern Panalytical) with a 565 nm filter and the data were analyzed with NanoSight software.
2.3. Stability of BRCs To assess the stability of BRCs, the nanoparticles and ssRNA mixture were incubated at 37°C with 10% fetal bovine serum-containing media (FBS; Gibco) for 30 min, 6 h, 12 h and
24 h. The RNA samples, including untreated BRCs, were resolved in 10% non-denaturing polyacrylamide gel electrophoresis (PAGE) gels at 85 V for 70 min in 1X TBE buffer (BioRad). The gels were then stained with 1 X GelRed (41003, Koma Biotech) in 1X TBE and analyzed under UV with a GelDoc EZ Imager (Bio-Rad). This experiment was carried out three times and the original images of the gels are shown in Figure 1F and Supplemental Figure 2. The band intensities were analyzed using the GelDoc software and plotted as shown in Figure 1G.
2.4. Cell lines and cell culture All cells were obtained from ATCC and were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS and maintained in a 5% CO2 incubator at 37°C.
2.5. Western blotting For western blotting analysis, tissues and cells were homogenized in lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, 0.5% Triton X-100, 1 mM EDTA) supplemented with protease inhibitors (Roche). The lysates were then centrifuged at 13,500 g for 30 min and the supernatants were recovered. About 30 µg of protein extracts were resolved by SDS-PAGE and analyzed by western blotting using primary antibodies against UBA6 [4], USE1 [5], Cyclin D (2978, Cell Signaling Technology), Cyclin E (4129, Cell Signaling Technology), Caspse-3 (9662, Cell Signaling Technology), p53 (sc-126, Santa Cruz Biotechnology), Bax (sc-7480, Santa Cruz Biotechnology), α-β-actin (sc-47778, Santa Cruz Biotechnology), and α-alpha-tubulin (ab7291, Abcam). This was followed by incubation with a corresponding IgG horseradish peroxidase-conjugated secondary antibody (ThermoFisher Scientific) and detection using enhanced chemiluminescence (Bio-Rad).
2.6. Apoptosis and cell cycle analysis Apoptosis analysis was carried out using an annexin V–FITC Apoptosis Detection Kit (Invitrogen). Briefly, cells (4 × 105) were exposed to 2 µg of scrambled control and USE1 BRC molecules for 48 h. The cells were then collected by centrifugation and resuspended in 500 µl of 1x binding buffer, followed by the addition of annexin V–FITC (5 µl) and PI (5 µl). After a short incubation at room temperature for 5 min in the dark, the cells were analyzed by FACS using a flow cytometer (FACSCalibur; BD Biosciences). The cells that stained positively for early apoptosis (annexin V–FITC-stained only) and for late apoptosis (annexin V–FITC- and PI-stained) were combined for analysis. Propidium iodide (PI) staining was used to analyze the DNA content and cell cycle distribution. After exposure to different concentrations of magnolol for 24 h, the cells were harvested and fixed in 70% ethanol, centrifuged (1500 rpm, 5 min), incubated with RNase (100 mg⁄mL) at 37°C for 30 min, and stained with PI (50 mg⁄mL in PBS). The cellular DNA content and cell cycle distribution were then analyzed by flow cytometry (FACSCalibur).
2.7. Cell proliferation assay For siRNA transient transfection assays, cells were seeded in 10 cm dishes at a density of 5 × 106 cells/dish and transfected with 20 nM siCDH17 or scrambled siRNA. After a 48 h incubation, the cells were disassociated and transferred to a 96-well plate at a density of 3000 cells/well, and then incubated for another 72 h. Cell viability was assayed using a Cell Counting Kit (CCK)-8 (Donjindo). For BRC transfection assays, the cells were seeded into 6 well plates at a density of 4 × 105cells/well and transfected with 2 µg USE 1 BRC or the scrambled BRC control. After 48 h of incubation, the cells were disassociated and transferred to 96-well plates at a density of 1000 cells/well, then incubated for another 0, 24, or 48 h.
Cell viability was assayed again using the CCK-8 kit. The absorbance (λ, 450 nm) of the resulting solution was measured with a spectrophotometer.
2.8. Scratch wound-healing assay For a scratch wound-healing assay, the cells were first seeded at 5 × 105 cells per well in 6well plates and incubated. Cell monolayers were then scratched with a sterile 200 µl pipette tip and washed with medium to remove detached cells. Cell migration was evaluated by measuring the differences in the wound areas on the monolayer.
2.9. Invasion assay To measure invasiveness, the cells were seeded at 1 × 105 cells per well in the upper chamber of a transwell insert (8-µm polycarbonate membrane; Corning) coated with 1–2 mg of matrigel (BD Biosciences) that was placed into a 24-well plate. The lower chamber was then filled with 800 µl DMEM supplemented with 10% fetal bovine serum and incubated for 36 h. Cells that invaded the matrigel to the underside of the membrane were fixed with 4% paraformaldehyde and stained with 0.5% crystal violet for 20 min. Cells that remained at the upper surface of the insert were removed with a cotton swab. The invading cells were counted in five random fields under a light microscope.
2.10. Colony formation assay To assess colony formation, cells were seeded at 1 × 103 cells per well in 6-well dishes and cultured in DMEM supplemented with FBS for 2 weeks. The cells were then fixed with 4% paraformaldehyde and stained with 0.5% crystal violet for 30 min.
2.11. Animals and tumor model
Female BALB/c nude mice (5 weeks old) were purchased from Orient Bio Inc. All animal studies were performed in accordance with the Institutional Ethics Committee and Institutional Animal Care Committee of University of Ulsan College of Medicine (2017-12281). A549 cells (1 × 107) were injected subcutaneously into the left flank of the mice. Tumor volumes (V) were then measured by V = L (w)2 × 0.5, where L is the length and w is the width of tumor.
2.12. In vivo therapeutic efficacy The antitumor effects of the USE1 BRC particles were investigated in lung cancer xenografts comprising A549 cells. When the tumor volumes reached a size of about 100 mm3, the mice were randomly split into 3 groups (n=5 per group). The animals were then anesthetized and administered intravenously with PBS, a scrambled BRC control, or USE1 BRC, respectively. The concentrations of the scrambled and USE1 BRC molecules were normalized to 5 µg in 0.1 mL PBS solution. BRC particles were injected intravenously three times in a week for 3 weeks. Tumor growth was recorded for 17 days. To further analyze any antitumor effects, the tumors were excised at 17 days after treatment and stained with hematoxylin and eosin (H&E). To visualize USE1 expression in the tumor tissues, immunohistochemistry with a USE1-specific antibody was conducted (final dilution 1:200). The slides were incubated overnight at 4°C and immunostaining was performed using a commercially available kit (Vectastain Universal Quick Kits and DAB, vector lab). A terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was carried out using a TdT-FragEL DNA fragmentation detection kit in accordance with the manufacturer’s instructions (Merck) to detect apoptotic cell death areas.
2.13. Biodistribution
Mice were injected via the tail vein with 100 µl of 15 µg SC BRC-Cy3, USE1 BRC-Cy3 or PBS. At 30 mins or 18 hours after injection (n= 3), the animals were euthanized and tissues were harvested including the heart, liver, lung, kidney and spleen. Each organ was imaged using the IVIS system (Perkin-Elmer). The average fluorescence intensity was determined for each tissue type using the same threshold settings after normalization with control organs.
2.14. Toxicity of the BRC molecules To assess the toxicity of the BRCs in vivo, additional mice were injected with 25ug SC BRC, USE1 BRC or PBS via the tail vein. At 24 hours after injection (n= 5), these animals were euthanized and the lung, liver, spleen and kidney were harvested and fixed in 10% formalin. The tissues were then paraffin embedded and H&E stained according to standard protocols. Each tissue was analyzed by a veterinary pathologist.
2.15. Biochemical assays Mouse blood samples were collected 24 hours after tail vein injection and then centrifuged. The serum obtained was stored at −20°C until analysis. The serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatinine (CREA), blood urea nitrogen (BUN), total protein (TP), and total bilirubin (TBIL) were then assayed using a Hitachi clinical analyzer 7180 (Hitachi). A complete blood count (CBC) test was performed using an Advia 2120i automated analyzer (Siemens).
3. Results 3.1. BRCs potentiate RNAi through their carefully engineered design By utilizing the recent advances in RNA nanotechnology, we fabricated BRCs using a complementary rolling circle transcription (cRCT) reaction to enable the efficient down-
regulation of USE1 expression [9, 13]. Based on previous findings, BRCs have doublestranded regions regularly spaced by a bubble region which enables the efficient release of siRNAs through cleavage by Dicer (Fig. 1A). The particle size was successfully engineered down to 350 nm in our previous study by manipulating the enzyme to template DNA ratios, but we aimed to further reduce this size in our present study. The self-assembly of the transcripts into 200 nm-sized particles was achieved by limiting the concentration of monomers in the cRCT reaction, whilst keeping the same 160:1 ratio of replicating enzyme to template DNA.
3.2. RNAs are packaged into BRCs with a highly porous structure and an adequate size for RNA delivery into tumor cells The highly porous surface morphology of our self-assembled BRC structure was revealed by scanning electron microscopy (SEM) (Fig. 1B). The high surface area of this structure facilitates easier access by the siRNA-releasing enzyme upon internalization into the target cells. Our average overall BRC size of 200 nm was further confirmed by atomic force microscopy (AFM; Fig. 1C), and the polydispersity index of these molecules (PDI; 0.150) measured by dynamic light scattering (DLS) indicated their monodispersity (Fig. 1D). It should also be noted that the sizes of these BRCs were within the favorable range for tumor site infiltration [14]. The increase in the hydrodynamic size indicated successful lipoplex formation upon introducing the transfection reagent, with the PDI remaining near 0.2, suggesting homogenous complexes were formed stably without aggregation (Supplemental Fig. 1). To confirm that the cRCT-induced nanostructure was generated from the RNA strands, a fluorescently labeled nucleotide triphosphate (cyanine 3-labeled UTP; cy3-UTP) was introduced to the cRCT reaction. The replicated RNA strands were then cy3 labeled by
incorporation of the cy3-UTP nucleotides by T7 RNA polymerase activity during the cRCT reaction. These fluorescently tagged nanostructures were evaluated by nanoparticle tracking analysis (NTA) which enables the measurement of nanoparticle size distribution and concentration. There was no significant difference found in the concentrations of the cy3labeled BRCs measured with the fluorescence and non-fluorescence mode of NTA, indicating that these molecules had been generated from cRCT reaction transcripts (Fig. 1E). Whilst the small size and porous structure of our BRC molecules facilitated a more optimal internalization into cancer cells and the ready access of the Dicer enzyme to release functional siRNAs, their compact structure also provided high physiological stability. This stability was assessed in the presence of 10% fetal bovine serum for different time periods i.e. from 30 min to 24 h (Fig. 1F, Supplemental Fig. 2). The results indicated that over 50% of the BRCs retained their molecular weights after 24 h of serum treatment, in contrast to the majority of an ssRNA mixture which was degraded within 30 min in the presence of serum (Fig. 1G).
3.3. A USE1 knockdown in both HeLa and A549 cells reduces cell proliferation and colony formation Since deregulated cell proliferation and enhanced colony formation are well established features of cancer [15-17], we tested the impact of USE1 silencing on A549 and HeLa cell growth in a CCK-8 assay. The results indicated in the first instance that USE1 BRCs were effective in suppressing the growth of these different cancer cell lines at the protein level (Fig. 2A, and Supplemental Fig. 3). The proliferative activities of the A549 and HeLa cells were found to gradually decrease when treated with USE1 BRC compared to the PBS and scrambled BRC controls (Fig. 2B, and Supplemental Fig. 4). We next evaluated the colony formation capacity of these cells under the same treatment conditions. As shown in Figure 2C,
the USE1 BRC-treated groups for both cell types had the lowest colony formation.
3.4. USE1 regulates the migration and invasion of HeLa and A549 cells A previous study has reported that USE1 is significantly associated with migration and invasiveness in lung cancer [5]. A scratch wound healing assay was performed to assess the migration-promoting ability of USE1 in A549 and HeLa cells, again using BRC particles to silence the gene (Fig. 3A and B). Both cell lines transfected with USE1 BRC molecules showed significantly reduced cell migration in a time-dependent manner, whereas no reduction was seen in the scrambled BRC control groups. In a matrigel invasion assay, the USE1 BRC-treated cells of both lines exhibited impaired invasiveness compared with the control groups (Fig. 3C). Taken together, these results indicated that the inhibition of USE1 impedes the migration and invasiveness of cancer cells.
3.5. USE1 silencing induces apoptosis and S phase cell cycle arrest in HeLa and A549 cells To confirm whether the proliferation suppression of HeLa and A549 cells caused by the RNAi silencing of USE1 was due to an apoptotic response, we analyzed these cells by flow cytometry using annexin V and PI staining. The apoptosis rate (annexin-V +/PI - and annexin-V+/PI +) of both USE1 BRC-treated cell lines at 48 h post transfection was indeed found to be elevated over the controls. The inhibition of USE1 significantly increased the apoptotic percentage in the HeLa and A549 cell populations by 48.8% and 37%, respectively, compared with the blank and scrambled BRC control cells (Fig. 4A). To further investigate the potential mechanisms by which USE1 silencing controls cancer cell growth, we examined the cell cycle of both the HeLa and A549 cell lines by flow cytometry following transfection with USE1 BRC particles. At 48 h post-transfection, the
proportion of USE1 BRC-treated HeLa cells in G1- and S-phase was reduced compared to the blank and scrambled controls. The percentage of cells in the sub G1-phase for the control, scrambled BRC-treated and USE1 BRC-treated HeLa cells was 0.3%, 2.8%, and 17.3%, respectively (Fig. 4B). The percentage of scrambled BRC-transfected A549 cells in S-phase was about 15-29%, whereas this was about 40% of the USE1 BRC-transfected A549 cells. At the same time, the number of USE1 BRC-treated A549 cells in the sub-G1 phase was higher than the two control groups (Fig. 4B). In addition, a downregulation of both cyclin D and E was detected (Fig. 4C). The protein levels of Bax and cleaved cas-3 were also found to be activated upon USE1 inhibition. Taken together, these results suggest that USE1 silencing suppresses the proliferation of cancer cells in vitro through cell cycle arrest and the induction of apoptosis.
3.6. Antitumor efficacy of USE1 BRC particles in an A549 tumor xenograft model The therapeutic effects of our USE1-targeting BRC particles were further examined in vivo using mice harboring A549 lung tumor xenografts. USE1 BRC particles, PBS, or a scrambled BRC control were injected intravenously into these tumors when the volume reached approximately 100 mm3. A total BRC amount of 0.025 mg/kg was administered three times a week for 3 weeks into the animals. Tumor growth was measured for 17 days after these treatments and the USE1 BRC injection resulted in a smaller tumor size compared with the controls (Fig. 5A). As shown in Figure 5B, the scrambled BRC control mice tumor volumes were comparable to those in the PBS control group. At day 10 post-treatment, the USE1targeted BRC group showed about a 70% tumor growth suppression compared with the two control groups (P < 0.05). To investigate the possible mechanisms of tumor growth inhibition in these experiments, USE1, p53, Bax and cleaved cas-3 were evaluated in the lung tumors by western blotting (Fig.
5D). The cellular concentrations of the USE1 proteins in the same tumor tissues were confirmed as being efficiently reduced by the administration of USE1 BRC. However, the expression levels of p53, Bax and cleaved cas-3, which are known signaling proteins for apoptotic cell death, were notably increased. The non-targeting scrambled BRC particles did not produce these effects under the same transfection conditions. To evaluate the antitumor activity of each treatment, histological examinations of the tumor tissue sections were performed using hematoxylin and eosin (H&E) staining and a TUNEL assay (Fig. 5E). The results revealed that cell death lesions and apoptotic tumor cells were localized in the tumors treated with USE1 BRC or scrambled BRC. However, the tumors in the mice treated with USE1 BRC showed increased cell death and larger apoptotic areas compared to the scrambled BRC control group. Furthermore, a reduced number of USE1 areas were detected in the USE1 BRC-treated group (Fig. 5E).
3.7. Biodistribution and toxicity of USE1 BRC molecules To investigate the biodistribution and toxicity of the USE1-targeting BRC particles in mice, USE1 BRC-Cy3 particles, PBS, or a scrambled BRC-Cy3 control were injected via the tail vein. After administration, BRC particle accumulation in different organs was detected using the IVIS system. A significant accumulation was observed in the kidney of USE1 BRC- and scrambled BRC-injected mice at 30 mins after injection (Fig. 6A). Interestingly, only USE1 BRC-Cy3 nanoparticles were observed in the kidney up to 18 hours after injection (Supplemental Fig. 5A). To assess the potential toxic effects of USE1 BRC molecules in the mice, blood biochemistry tests were conducted but showed that no significant differences between the groups (Fig. 6B). Subsequent histological analysis showed that the USE1 BRCs did not cause significant injury to the liver, kidney, spleen or lung tissues (Fig. 6B). These findings
indicated no induction of toxicity by USE1 BRC in vivo.
4. Discussion Our previous study implicated USE1 in lung cancer development and we reported that its overexpression promoted the proliferation, migration, and invasion of lung tumor cells [5]. USE1 was thus considered to be a molecular regulator of tumor growth in lung cancer. It is well known that most cancer types are defined by augmented cell proliferation and a reduced apoptotic response. Identifying molecular regulators of tumor cell growth is therefore an important part of cancer research. We have here presented the potency of BRCs in silencing USE1 via RNAi and assessed the effects of this treatment in the HeLa cervical cancer and A549 lung cancer cell lines to assess its antitumor efficacy. Our present results clearly indicate that the knockdown of USE1 using targeted BRCs significantly suppresses tumor cell growth both in vitro and in vivo. Mechanistically, we demonstrated an induction of S-phase cell cycle arrest and an apoptotic response through the p53 signaling pathway in cells transfected with USE1 BRCs. BRC control molecules had no toxic effects in the cells. Our findings therefore indicate that these molecules have potential clinical application as a new form of cancer therapy. Apoptosis is a well-known process for eliminating unhealthy cells [18] and is triggered by two distinct signaling pathways [19-22]. One of the major apoptotic pathways in cancer cells occurs in mitochondria [23] and we observed this process in A549 and HeLa cells after transfection of USE1 BRCs, with the consequent decrease in cell proliferation. In further functional studies, we assayed the levels of p53 and Bax, a pro-apoptotic factor [24], and also caspase-3, an apoptotic effector protein [25-29]. The USE1 BRC knockdown significantly augmented p53 and Bax expression. The caspases are another important activating component of p53-mediated apoptosis [30] and caspase-3 was found in our experiments to be
expressed at higher levels following treatment with USE1 BRCs. Cell cycle arrest is another pathway that can lead to apoptosis and is commonly deregulated in cancer [31, 32]. Consistently, the USE1 BRCs caused an S-phase arrest in both A549 and HeLa cells. In support of these findings, the critical regulator of S-phase arrest, cyclin E [33-35], along with the other crucial cell cycle factor cyclin D1, were found to be clearly suppressed by USE1 BRC transfection, indicating that the downstream effectors of USE1 are these cyclins. We employed an A549 xenograft model system to investigate whether the effects of the USE1 BRCs in vitro would be reproduced in vivo. The mice treated with USE1 BRCs showed a greater reduction of tumor growth than either the scrambled BRC or PBS control groups. Moreover, H&E staining and TUNEL assays of the USE1-suppressed A549 tumor sites revealed regions of necrosis and apoptosis. Of note, a lower expression of USE1 was confirmed in the USE1 BRC group. These findings were consistent with the cell cycle arrest and apoptosis caused by USE1 silencing in vitro. Moreover, when we further analyzed the key apoptotic factors p53, Bax, and caspase-3, we observed higher expression levels in tumor tissues in which USE1 had been silenced. In conclusion, USE1 targeting by BRCs in both cervical and lung cancer cells in vitro, and in A549 lung cancer xenografts in vivo, shows promise as an anti-cancer therapeutic strategy. Mechanistically, the suppression of cancer cell proliferation resulting from the USE1 knockdown was caused by a cell cycle arrest in S-phase and an enhanced apoptotic response. We propose that BRC-based therapies may prove to be effective in the future against various solid cancers.
AUTHOR INFORMATION Corresponding authors email: [email protected] and [email protected]
Conflict of interest declaration The authors declare no competing financial or other interests in relation to this article.
Acknowledgements This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (NRF-2019R1A2C2084181), and also supported by the Bio & Medical Technology Development Program of the National Research Foundation (NRF) funded by the Ministry of Science & ICT (2016M3A9C6917402).
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Table 1. DNA sequences used to generate USE1-targeting BRCs. /5Phos/ indicates 5’ phosphorylation. The primer binding, USE1 pre-siRNA, and non-targeting pre-siRNA regions are indicated in purple, red, and orange, respectively.
Name USE1 siRNA targeting region in the mRNA Primer for the T7 promoter region
Sequence (5’ → 3’) (mRNA) GGG AAA GUC UGC UUG AGU A TAA TAC GAC TCA CTA TAG GGA T
USE1 pre-siRNA sense strand
/5Phos/ ATA GTG AGT CGT ATT AAA GGG AAA GTC TGC TTG AGT ATT GCT GGA TGA AGG ACG GTC GAA CGC AAG GGA AAG TCT GCT TGA GTA TTA TCC CT
USE1 pre-siRNA antisense strand
/5Phos/ ATA GTG AGT CGT ATT AAA TAC TCA AGC AGA CTT TCC CTT CTT AGG CTG GAC AAC AAC CAT CTA AAT ACT CAA GCA GAC TTT CCC TTA TCC CT
Non-targeting presiRNA sense strand
/5Phos/ ATA GTG AGT CGT ATT AAA GTA TCT CTT CAT AGC CTT ATT GTA TCT CTT CAT AGC CTT AAA GTA TCT CTT CAT AGC CTT ATT ATC CCT
Non-targeting presiRNA antisense strand
/5Phos/ ATA GTG AGT CGT ATT AAA TAA GGC TAT GAA GAG ATA CTT GTA TCT CTT CAT AGC CTT AAA TAA GGC TAT GAA GAG ATA CTT ATC CCT
Figure Legends
Fig. 1. (A) Schematic illustration of the synthesis of USE1-targeting BRCs. (B) Low and high magnification (inset) SEM images of the BRCs, revealing their spherical shape and porous surface. (C) 3D reconstructed AFM image of the BRCs suggesting that their overall zdimensional sizes are ~200 nm. (D) Size distribution and polydispersity index of the BRCs analyzed by DLS, indicating their monodispersity. (E) Concentrations of cy3-labeled BRCs measured by NTA with or without a 565 nm filter (student’s t test; n.s., not significant). (F) Stability analysis of ssRNA and BRCs in the presence of 10% FBS, analyzed by nondenaturing PAGE. (G) Band intensity analysis of the polyacrylamide gels in panel (F) and in Supplemental Figure 2.
Fig. 2. A USE1 knockdown reduces the proliferation and colony formation capacity of HeLa and A549 cells. (A) USE1 protein levels in HeLa and A549 cells upon transfection with 2 µg USE1 BRC for 48 h, analyzed by western blot. Scrambled BRC transfected cancer cells (2 µg) and blank cells were used as controls. (B) Effects of USE1 silencing on HeLa and A549 proliferation, detected by CCK-8 assay. Results represent the means ± SE (n = 4); ***P < 0.001. (C) Effects of USE1 silencing on the colony formation ability of HeLa and A549 cells, tested using a plate colony assay. The relative colony formation efficiency of the blank cells was set as 1. Each column represents the mean value from triplicate experiments in each group. Data are the mean ± SE; *P < 0.001.
Fig. 3. USE1 regulates the migration and invasion of HeLa and A549 cells. (A, B) The migration ability of HeLa and A549 cells was assessed using an in vitro scratch wound healing assay. Images were taken 0, 24, and 48 h after wounding of the cell monolayers. (C, D) The invasiveness of the HeLa and A549 cells was evaluated using a Transwell assay, and images were captured at 48 h after incubation of the cells in a matrigel-precoated Transwell chamber (original magnification, × 200); ***P <0.001 compared with scrambled BRC-treated cancer cells.
Fig. 4. A USE1 knockdown in HeLa and A549 cells induces apoptosis and S-phase cell cycle arrest. (A) Apoptotic cells was detected by annexin V and PI double-staining. A total of 2 µg of USE1 BRC was transfected into the cells for 48 h. (B) The cell cycle distribution was assessed by flow cytometry at 48 h post-treatment. (C) Effect of USE1 silencing on the expression of cyclin D1, cyclin E, Bax, cleaved cas-3 and USE1. β-actin was used as an internal loading control.
Fig. 5. Antitumor efficacy of USE1 BRC particles in an A549 xenograft model. (A) Dissected tumors in each treatment group are shown at 17 days post-treatment. The A549 xenografts in the mice were injected intravenously with PBS, scrambled BRC or USE1 BRC three times per week for three weeks. (B) Tumor growth was measured over 17 days postinjection by assessing the tumor volumes using the formula: L (w)2 × 0.5, where L is the largest and w is the smallest diameter. Data represent the mean ± SE (n = 5); **P < 0.01, ***P < 0.001 versus the Control and Scrambled BRC groups. (C) Changes in the mouse body weights during the aforementioned treatments. (D) USE1 silencing is associated with the activation of p53, Bax and caspase-3 in the A549 xenograft tumors. (E) Histological analysis of xenograft tumor tissues treated with PBS, scrambled BRC, or USE1 BRC. Tumor tissues stained with H&E, a USE1-specific antibody, and TUNEL were viewed under a light microscope. Original magnifications in all three cases, × 100. Scale bar, 10 µm.
Fig. 6. Ex vivo optical images and biochemical toxicity of USE1 BRC in mice. (A) Biodistribution of USE1 BRC particles in the mice. The mice were injected intravenously with PBS, scrambled BRC-Cy3 or USE1 BRC-Cy3. The biodistribution of the BRCs in selected organs (liver, lung, heart, spleen and kidney) was analyzed using the IVIS system at 30 min after injection; n=3 in each group. (B) Effect of USE1 BRC particles injection in mice. H&E staining, complete blood count (CBC) and various biochemical parameters were evaluated, and data were expressed as a median ± standard deviation; n=5 in each group. The units followed by WBC (103/µL), RBC (106/ µL), HGB (g/dL), PLT (103/µL), AST (U/L), ALT (U/L), BUN (mg/dL), TP (g/dL), TBIL (mg/dL) and CREA (mg/dL). Original magnifications in all three cases, × 100. Scale bar, 10µm.
Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: