Abstract
An in vitro-transcribed RNA aptamer (trans-RA16) that targets non-small cell lung cancer (NSCLC) was previously identified through in vivo SELEX. Trans-RA16 can specifically target and inhibit human NCI-H460 cells in vitro and xenograft tumors in vivo. Here, in a follow-up study, we obtained a chemically-synthesized version of this RNA aptamer (syn-RA16) and a truncated form, and compared them to trans-RA16 for abilities to target and inhibit NCI-H460 cells. The syn-RA16, preferred for drug development, was by design to differ from trans-RA16 in the extents of RNA modifications by biotin, which may affect RA16’s anti-tumor effects. We observed aptamer binding to NCI-H460 cells with KD values of 24.75 ± 2.28 nM and 12.14 ± 1.46 nM for syn-RA16 and trans-RA16, respectively. Similar to trans-RA16, syn-RA16 was capable of inhibiting NCI-H460 cell viability in a dose-dependent manner. IC50 values were 118.4 nM (n = 4) for syn-RA16 and 105.7 nM (n = 4) for trans-RA16. Further studies using syn-RA16 demonstrated its internalization into NCI-H460 cells and inhibition of NCI-H460 cell growth. Moreover, in vivo imaging demonstrated the gradual accumulation of both syn-RA16 and trans-RA16 at the grafted tumor site, and qRT-PCR showed high retention of syn-RA16 in tumor tissues. In addition, a truncated fragment of trans-RA16 (S3) was identified, which exhibited binding affinity for NCI-H460 cells with a KD value of 63.20 ± 0.91 nM and inhibited NCI-H460 cell growth by 39.32 ± 3.25% at 150 nM. These features of the syn-RA16 and S3 aptamers should facilitate the development of a novel diagnostic or treatment approach for NSCLC in clinical settings.
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Introduction
Lung cancer is the leading cause of cancer-related mortality in men and the second leading cause in women worldwide1,2,3. Thus far, the two main types of lung cancer are non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). NSCLC accounts for 85–90% of all lung cancer cases4. Chemotherapy is the primary treatment of choice for NSCLC; however, there are side effects such as gastrointestinal distress, organ damage, and even death5,6. In comparison with SCLC, NSCLC is relatively insensitive to chemotherapy, thus increasing the risk of mortality. In order to increase the survival rate of patients with NSCLC, there has been great interest in developing new targeted therapies and combination therapies.
Recently, the specific targeting of monoclonal antibodies and Chimeric Antigen Receptor (CAR)-T cells have contributed to the treatment of cancers including NSCLC. However, the high cost and sophisticated techniques required for manufacturing these bio-products are major challenges in clinical applications7,8,9,10,11. Aptamers are a class of single-stranded oligonucleotides (RNAs or ssDNAs) that can serve as ligands that recognize and bind to their targets with specificity and high affinity12. Typically, specific aptamers are generated by a process known as Systematic Evolution of Ligands by Exponential Enrichment (SELEX)13,14. Since the invention of SELEX in the 1990s, specific aptamers for various targets have been identified15,16,17.
A previous study by our group demonstrated the potential of a NSCLC-specific RNA aptamer selected via in vivo SELEX18. The aptamer, named RA16, was capable of binding to and inhibiting NSCLC human large cell lung cancer cell line NCI-H460 cells in vitro and in vivo, which may be applied to tumor imaging technique and targeted therapies. A major advantage of RNA aptamers is that they can be chemically synthesized for use in diagnosis, treatment and biomarker discovery. Therefore, the binding and inhibitory activity of the synthesized RA16 (syn-RA16), as well as the potential mechanisms should be further investigated. Furthermore, a smaller aptamer size could facilitate large-scale chemical synthesis and would be beneficial for clinical applications.
Here, we conducted a sequential study of the syn-RA16 and truncated aptamers specifically targeted and directly inhibited towards NCI-H460 cells in vitro and in vivo. We also demonstrated the potential tentative mechanism for syn-RA16 internalization and intracellular signaling mechanism.
Results
Specificity and Affinity of syn-RA16 in vitro
We previously reported that the NSCLC-specific RA16 selected via in vivo SELEX could bind to NSCLC NCI-H460 cells in vitro. To further confirm the specificity of the selected syn-RA16 aptamer, its binding activity was evaluated and compared with that of trans-RA16 in vitro. The syn-RA16 or biotin-labeled trans-RA16 aptamer was incubated with NCI-H460 cells, NSCLC (human lung adenocarcinoma cell line SPC-A1 cells), or control cells including human embryonic kidney-293T (HEK-293T) cells, human cervical carcinoma cell line (HeLa cells), and human normal lung cell line (BEAS-2B cells). Scrambled RNA (SCAP) served as the negative control. Fluorescence was detected using streptavidin-Alexa Fluor 488, and the cells were imaged under a microscope. Fluorescent binding was observed for NCI-H460 cells, but not SPC-A1, HEK-293T, HeLa, and BEAS-2B cells (Fig. 1A,B), suggesting that syn-RA16, similar to trans-RA16, demonstrated specific binding to NCI-H460 cells in vitro.
To assess the binding affinity of syn-RA16 and compare it with trans-RA16, flow cytometry was performed. As shown in Fig. 1C, when NCI-H460 cells were incubated with biotin-labeled RNA molecules, syn-RA16, similar to trans-RA16, bound to most of the cells and exhibited a clear fluorescence shift detected by streptavidin-PE, thus indicating the specific binding between syn-RA16 and NCI-H460 cells. There was no noticeable fluorescence shift for the negative control (Fig. 1C). We then determined the binding affinity of syn-RA16 and trans-RA16 for NCI-H460 cells by measuring the equilibrium dissociation constant (KD). Analysis of the fitted lines revealed KD values of 24.75 ± 2.28 nM and 12.14 ± 1.46 nM for syn-RA16 and trans-RA16, respectively (Fig. 1D), demonstrating the high affinity of the aptamers for NCI-H460 cells.
Specific binding mediated internalization of aptamer RA16
As reported, most cell-binding aptamers rely on cell membrane biomarkers for recognition and internalization through endocytosis19,20,21. To further investigate the time course of RA16 and potential mechanism for aptamer internalized, we performed time course study to monitor aptamer syn-RA16 internalization with different incubation time points.
As shown in Fig. 2A, syn-RA16 gradually entered NCI-H460 cells, and binding of syn-RA16- NCI-H460 cells was detectable after 1 h incubation. When the incubation time was increased to 2 h, RA16 gradually entered the cell and signaled at the most in cytoplasm. Colocalization of aptamer RA16 and lysotracker revealed that the most of the aptamer entered cells through endocytosis (Fig. 2B). After 4 h incubation, the syn-RA16 further accumulated in the cytoplasm.
To further quantify time-depending internalization of RA16 to NCI-H460 cells, NCI-H460 cells were incubated with syn-RA16 or SCAP in serum containing medium for different incubation time. The relative aptamer recovery levels were further detected by quantitative RT-PCR. The internalized aptamer kinetics were shown in Fig. 2C. Consistent with the imaging data, SCAP showed low internalization in NCI-H460 cells. On the contrast, syn-RA16 demonstrated a significantly strong internalization. As time increasing, syn-RA16 gradually internalized and reached at the maximum after 4 h incubation.
The results indicated that aptamer RA16 entered NCI-H460 cells through receptor-mediated endocytosis. The specific binding triggered the aptamer internalized, migrated, and finally accumulated in the cytoplasm. The possible mechanism of aptamer binding and signaling courses is shown in Fig. 2D.
Inhibition of cell growth in vitro
Trans-RA16 has been demonstrated previously to inhibit NCI-H460 cell growth18. Therefore, we performed cell viability assays to assess the inhibitory activity of syn-RA16. As shown in Fig. 3A, the inhibition rates of NCI-H460 cell growth at various concentrations by syn-RA16 and trans-RA16 were almost similar. Notably, syn-RA16 suppressed NCI-H460 cells by 84.5%, whereas trans-RA16 suppressed the cells by 86.8% at 300 nM. NCI-H460 cells treated with both trans-RA16 and syn-RA16 exhibited an apoptotic phenotype (Fig. 3B). Furthermore, analysis of the inhibition rate with a concentration series of RA16 revealed IC50 values of 118.4 nM and 105.7 nM for syn-RA16 and trans-RA16, respectively (Fig. 3C). Interestingly, both syn-RA16 and trans-RA16 did not have an inhibitory effect on HeLa cells at 600 nM (Fig. 3D).
Tumor-targeting efficacy in vivo
After evaluating the specificity and inhibitory activity of syn-RA16 in vitro, we further performed an in vivo tumor imaging assay to investigate the targeting activity of syn-RA16 in vivo. Cy5.5-labeled syn-RA16 or trans-RA16 was injected into NCI-H460 tumor-bearing mice to track the movements of the specific RA16 molecules in vivo. SCAP was used as the control. At 0.5 h, syn-RA16 and trans-RA16 reached the tumor site with a weak fluorescence signal. As time increased, RA16 aptamers gradually accumulated at the tumor site with a strong fluorescence signal at 2 h. Notably, no fluorescence signal was observed in the tumors of mice injected with SCAP (Fig. 4A). Subsequently, we extracted the tumors for imaging; fluorescence signals were much higher in mice injected with syn-RA16 or trans-RA16 than in those injected with SCAP (Fig. 4B), demonstrating the specific target binding of syn-RA16 in vivo.
An in vivo trap assay with quantitative reverse transcription polymerase chain reaction (qRT-PCR) was also performed to evaluate the distribution of syn-RA16 in vivo. NCI-H460 tumor-bearing mice were injected with syn-RA16, trans-RA16, or SCAP. Total RNA was then collected from various tissues for RNA quantification (normalized to mouse 18S RNA). The graph of RNA distribution is shown in Fig. 4C (n = 4). The level of syn-RA16 was significantly higher (50- to 1000-fold) in tumor tissues than in other tissues such as liver, kidney, heart, and lung tissues. Similar results were observed for trans-RA16. Moreover, the level of RNA molecules in the tumor tissues was significantly different between the RA16 groups and SCAP group. As shown in Fig. 4C, trapped RA16 was 100-fold higher than trapped SCAP at the tumor site, indicating that the entrapment of syn-RA16 in tumor tissues may be attributed to its specific binding in vivo. Overall, the results demonstrated the tumor-specific targeting activity of syn-RA16 and trans-RA16 both in vitro and in vivo.
Cell binding and inhibitory activity of a truncated aptamer
In order to minimize the functional motif and facilitate large-scale chemical synthesis, RA16 aptamers were truncated into three smaller parts (S1 containing 40 random nucleotides, S2 containing 40 nucleotides with the 3′-end, and S3 containing the 5′-end with 40 random nucleotides). The potential secondary structures of RA16 and three smaller fragments were predicted by Mfold software (http://unafold.rna.albany.edu/?q=mfold), as shown in Fig. 5A22.
Binding activity was assessed by flow cytometry. As shown in Figs. 5B and S1, S2, and SCAP (negative control) were unable to bind to the target cells. However, S3, similar to RA16, exhibited binding affinity for NCI-H460 cells. The binding affinity (KD) was 63.20 ± 0.91 nM (Fig. 5C).
In addition, the inhibitory activity of S3 was further determined. As shown in Fig. 5D,E, similar to RA16, NCI-H460 cells treated with S3 exhibited an apoptotic phenotype. S3 inhibited H460 cell growth by 39.32 ± 3.25% at 150 nM, while RA16 suppressed the cells by 61.79 ± 3.27%. These results indicated that S3 containing the 5′-end with 40 random nucleotides of RA16 retained cell-binding and inhibitory activity.
Discussion
NSCLC is one of the leading causes of cancer-related deaths worldwide4. Although there are severe side effects, traditional treatment strategies, such as chemotherapy, remain the primary treatment of choice in clinical settings5,6.
There have been various efforts to develop novel targeted therapeutics and overcome the drawbacks of chemotherapy. In comparison with chemotherapy, small molecules such as epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have been reported to play a major role in enhancing the survival rate of patients with NSCLC and decreasing toxicity23. However, studies have reported that EGFR TKIs did not affect patients harboring wild-type EGFR6,24. Furthermore, resistance can invariably occur23,24. Monoclonal antibodies such as cetuximab and bevacizumab may be effective in attenuating the progression of lung cancer7,8,10. However, they face similar resistance problems23. In comparison with tumor-targeting monoclonal antibodies, aptamers have several advantages such as (i) production via chemical synthesis, (ii) no or low immunogenicity, (iii) a smaller molecular size, (iv) efficient biological compartments penetration, and (v) ease of conjugation to various nanomaterials25,26,27,28. Previous studies have revealed that specific cancer aptamers are useful for in vitro tumor diagnosis, in vivo tumor imaging technique29,30,31,32, and targeted tumor therapy2,20,33,34,35.
Owing to their smaller size, specific binding, and tissue-penetration activity, RNA aptamers are considered as ideal agents for cancer diagnosis and cancer-targeted therapy. Aptamers specific for cancer-related proteins including vascular endothelial growth factor (VEGF), EGFR, mucin 1 (MUC1), and p53 have been identified15,31,32,36. Previous studies on targeted chemotherapeutic delivery and tumor imaging have demonstrated the potential of aptamers for targeted treatment and cancer diagnosis29,30,33,37,38,39,40. Recently, an NSCLC-specific RNA aptamer was selected via in vivo SELEX18. Binding activity of RA16 to NSCLC cell line (NCI-H1299, SPC-A1, and NCI-H1650 cells), as well as non-NSCLC (HeLa and 293 T cells) were detected respectively, which demonstrated high specificity and affinity towards specific NSCLC tumors. A major advantage of aptamers is the ease of chemical synthesis. Giving synthetic RNA aptamers have a more uniform and highly purified consistent stable structure, the syn-RA16 could easily be adopted for large-scale and cost-efficient production in clinical application. In addition, the syn-RA16 would be beneficial for further modifications such as incorporation of 2′-F dCTP/UTP and 5′-PEGylation, as well chemical adducting and manufacturing18. Obviously, the advantages of synthesized aptamers would be more feasible for applications of the clinic.
In this study, we evaluated the specific target binding and direct inhibitory activity of syn-RA16. As we tested and determined the binding affinity in the preliminary study, most of the non-NSCLC cell line showed no or little binding towards RA16, even at high concentration of syn-RA16 at 600 nM. It is our understanding that it’s impossible to determine the dissociation constant in lung normal cell lines and in non-NSCLC cell lines. We only determine the dissociation constant in NSCLC H460 cells. Although nucleotide sequences of syn-RA16 and transcribed RA16 are basically the same, syn-RA16 was produced by Dharmacon (GE Healthcare, Lafayette, CO), and trans-RA16 was transcribed from a DNA template in vitro. The main difference between syn-RA16 and trans-RA16 are their labeling status and purity. The affinity of syn-RA16 was slightly lower than that of trans-RA16 as demonstrated by KD determination assay. This result may be attributed to the presence of only one biotin-labeled site in syn-RA16. On the other hand, additional biotin-labeled sites could be incorporated during the in vitro transcription process, resulting in a more sensitive fluorescence signal produced by trans-RA16. However, inhibitory activity was almost similar based on IC50 values for both syn-RA16 and trans-RA16 (118.4 nM vs. 105.7 nM). We also assessed the specific targeting of syn-RA16 by in vivo tumor imaging and qRT-PCR. Both syn-RA16 and trans-RA16 showed high retention in NCI-H460 tumor tissues in vivo. In fact, a more uniform flow binding profile with syn-RA16 was observed (Fig. 1C), indicating that the preparation of syn-RA16 was more purified than the trans-RA16. This result is consistent with a more tumor and lung bindings for syn-RA16 because with the same molar unit preparations of syn-RA16 and trans-RA16, the more active syn-RA16 aptamers were binding to tumor or lung tissues of NSCLC (Fig. 4C).
In addition, we further investigated the potential mechanism for aptamer binding and growth-inhibiting effects. Based on the time course and cell cycle analysis studies, it is our tentative hypothesis that aptamer RA16 firstly bound to the cell and triggered internalization, followed by further migration and accumulation in the cytoplasm. The internalized aptamer RA16 may regulate some intracellular pathways of NCI-H460 cells, such as, interfering the processes of the protein transcribing or translating in the cell cytoplasm. As a result, these effects may inhibit NCI-H460 cell growth. The tentative mechanism of the aptamer in cell binding and inhibition was proposed as shown in Fig. 2D. Moreover, our preliminary data (unpublished) showed that the target of RA16 is most likely a protein-related component.
On the other hand, a truncated fragment of RA16 (S3) was found to exhibit cell binding and inhibitory activity. Notably, more than three structures for S1 were predicted and only one possible structure for RA16 and S3 was predicted (as shown in Fig. 5A). S3 retained the secondary structure of RA16 at the 5′ end, and the other two truncated fragments (potentially folded into other structures) did not bind to target cells, indicating that the secondary/tertiary structure plays a major role in aptamer activity. The 5′-end structure of RA16 could be critical for specific binding and intracellular signaling. This region may induce aptamer internalization and lead to intracellular signaling for cell growth inhibition. However, the affinity (KD) and inhibitory effect of S3 were slightly lower than that of the full-length aptamer (63.20 ± 0.91 nM vs. 12.14 ± 1.46 nM; 39.32 ± 3.25% vs. 61.79 ± 3.27%) which is largely consistent with the truncation process, indicating that the 3′-end of the aptamer may also contribute to structure folding and target binding41,42.
In conclusion, we conducted a sequential study of the anti-NSCLC aptamer RA16, which can be chemically synthesized. In this study, syn-RA16 demonstrated specificity and high affinity for NSCLC NCI-H460 cells in vitro and in vivo. Notably, we demonstrated the tentative mechanism for syn-RA16 binding and intracellular signaling. In addition, the truncated aptamer (S3) exhibited cell binding and inhibitory activity, indicating that this aptamer could be further truncated and modified. The syn-RA16 and truncated aptamer could contribute to the identification of potential targets and elucidation of molecular mechanisms, which would be beneficial for future applications of the aptamer as a drug or diagnostic reagent for NSCLC.
Materials and Methods
Oligonucleotides
Full-length 2′-fluoropyrimidine RA16 aptamer (syn-RA16) was synthesized by Dharmacon (GE Healthcare, Lafayette, CO).
Syn-RA16: 5′-Biotin-GGGAGAGAACAAUGACCUGCGGUGCCAAGCCGUCGGGUUAUGUUGAUCUCCACAAGGACGAGUGCAUUGCAUCACGUCAGUAG-NH2-3′.
The DNA template for transcription and primers were synthesized by Integrated DNA Technologies (IDT, Coralville, IA).
DNA template: 5′-CACTAATACGACTCACTATAGGGAGAGAACAATGACCT GCGGTGCCAAGCCGTCGGGTTATGTTGATCTCCTCAAGGACGAGTGCATTGCATCACGTCAGTAG-3′. Forward primer: 5′-CACTAATACGACTCACTATAGGGAGAGAACAATG-3′. Reverse primer: 5′-CTACTGACGTGATGCAATGCACTC-3′.
Cells
NCI-H460, HEK-293T, SPC-A1, HeLa, BEAS-2B, and other cell lines were purchased from the American Type Culture Collection (Manassas, VA). Tumor cell lines were cultured in Roswell Park Memorial Institute 1640 medium (Thermo Fisher Scientific, Rockford, IL). HEK-293T, HeLa, and BEAS-2B cells were cultured in Dulbecco’s modified Eagle’s medium (Thermo Fisher Scientific, Rockford, IL) containing 10% (v/v) fetal bovine serum, GlutaMax, and 100 U/mL penicillin-streptomycin in an incubator (Thermo Fisher Scientific, Rockford, IL) at 37 °C, 5% CO2. Cells were sub-cultured approximately every 2 days at 80% confluence using 0.25% (w/v) trypsin (Thermo Fisher Scientific, Rockford, IL) at a split ratio of 1:3.
Animals
Female BALB/c nude mice were purchased from SLRC Laboratory Animal Center (Shanghai, China). All animal studies were performed in accordance with the Guide for Care and Use of Laboratory Animals, Soochow University. All animal experimental protocols were approved by the Soochow University Institutional Animal Care and Use Committee (IACUC Permit Number SYXK (Su) 2015-0105). The nude mice were injected with 2 × 106 NCI-H460 cells subcutaneously to establish a tumor-bearing mouse model.
Biotin labeling of transcribed aptamers
The DNA template of RA16 aptamers was in vitro transcribed into RNA in a reaction mixture consisting of 10× transcription buffer (400 mM Tris-Cl, 80 mM MgCl2, and 20 mM spermidine), 10 mM dithiothreitol, 20 U T7 mutant (Y639F) RNA polymerase, 10 mM ATP, 10 mM GTP (Sangon Technologies, Shanghai, China), 10 mM 2′-F-dCTP/UTP (TriLink Biotechnologies, San Diego, CA), 2 mM 16-Biotin-UTP (Sigma-Aldrich, St. Louis, MO), 20 U RiboLock RNase Inhibitor (Thermo Fisher Scientific, Rockford, IL), and 0.05 U inorganic pyrophosphatase (Thermo Fisher Scientific, Rockford, IL). The resulting reaction mixture was treated with 2 μL DNase I (5 U/μL, RNase-free; TaKaRa, Dalian, China) at 37 °C for 1 h, followed by phenol-chloroform extraction. RNA pellets were suspended in RNA refolding buffer (10 mM HEPES pH 7.4, 50 mM NaCl, 1 mM CaCl2,1 mM MgCl2, and 2.7 mM KCl), followed by refolding at 90 °C for 3 min and slowly cooling to room temperature20.
Fluorescent labeling of aptamers
The DNA template was transcribed by substituting 16-Biotin-UTP for aminoallyl-dUTP (TriLink Biotechnologies, San Diego, CA) to generate aminated RNA. Both trans-RA16 and syn-RA16 were suspended in 0.1 M NaHCO3 (pH 8.3) and incubated with NHS-Cy5.5 (GE Healthcare, Marlborough, MA)43. After 2 h of reaction at room temperature, 10 mM Tris was added to neutralize excess fluorescent dye. Then, the mixture was filtered using Amicon YM-10 filter (Merck Millipore, Darmstadt, Germany) to generate fluorescently labeled RA16.
Cell binding assay
NCI-H460, HEK-293T, SPC-A1, HeLa, and BEAS-2B cells were grown to 70% confluence in 24-well plates. After washing with Dulbecco’s phosphate-buffered saline (DPBS; Thermo Fisher Scientific, Rockford, IL) twice, the cells were incubated with 200 nM biotin-labeled aptamers in binding buffer (RNA refolding buffer containing 1% bovine serum albumin and 1.0 μg/mL tRNA) for 1 h at 37 °C36. Next, the cells were washed with RNA refolding buffer and stained with streptavidin-Alexa Fluor 488 (Thermo Fisher Scientific, Rockford, IL) according to the manufacturer’s protocol. The nucleus was stained with Hoechst 33342 (Thermo Fisher Scientific, Rockford, IL) at 37 °C for 5 min and washed with DPBS twice. Then, the cells were imaged under a microscope (Olympus, Tokyo, Japan).
Affinity determination
NCI-H460 cells were digested with 0.25% trypsin. Next, 1 × 105 cells were incubated with a concentration series (0 to 200 nM) of biotin-labeled syn-RA16 or trans-RA16 in 100 μL binding buffer for 1 h at 37 °C. The cells were then washed with DPBS twice and stained with streptavidin-PE (BioLegend, San Diego, CA) according to the manufacturer’s protocol44. After washing and re-suspension in fluorescence-activated cell sorting (FACS) buffer (BD Biosciences, San Diego, CA), the cells were subjected to flow cytometry analysis. A total of 10,000 events were acquired for each sample using the FACSVerse® system (Becton Dickinson, Franklin Lakes, NJ), and data were obtained and analyzed by FlowJo® software (version X 10.0, https://www.flowjo.com/). The mean fluorescence intensity (MFI) of each sample was determined to calculate the dissociation constant (KD) between aptamers and NCI-H460 cells by linear fitting according to the equation 1/F = (KD + [L])/Bmax × [L] (where F = fluorescence intensity and [L] = concentration of aptamers)44,45.
Time course study
NCI-H460 cells were grown to 70% confluence on coverslips. The coverslips were washed with DPBS twice, and incubated with 200 nM biotin-labeled aptamers in binding buffer for 0.5 h, 1 h, 2 h, and 4 h at 37 °C. For the cells of colocalization, 100 nM LysoTracker™ Deep Red (Thermo Fisher Scientific, Rockford, IL) was added to the culture medium for imaging study. The coverslips were then fixed with 4% paraformaldehyde for 10 min and washed twice with DPBS. Cells were then stained with streptavidin-Alexa Fluor 488 according to the manufacturer’s protocol. The nucleus was stained with Hoechst 33342 at 37 °C for 5 min and washed with DPBS twice. The cells were then mounted and imaged under a confocal microscope (Olympus, Tokyo, Japan).
Time course study with qRT-PCR
NCI-H460 cells were seeded in 24-well plates at 1 × 105 cells per well overnight at 37 °C. The medium was then removed, and the cells were treated with 300 nM syn-RA16 or scrambled RNA (SCAP) in fresh medium for series incubation time (0, 15, 30, 60, 120, 240, 480, and 960 min). After incubation, cells were rinsed three times with DPBS, and then collected for RNA extraction using TRIzol reagent (Thermo Fisher Scientific, Rockford, IL) according to the manufacturer’s protocol. The extracted total RNAs were first treated with DNase I to eliminate DNA contamination and quantified using One Drop Spectrophotometry (Hong Kong, China). Next, 500 ng of DNase I-treated RNA was reverse-transcribed into DNA using M-MLV transcriptase (TaKaRa, Dalian, China). Real time PCR was performed with aptamer primers and Power SYBR Green Master Mix (Life Technologies) according to the manufacturer’s protocol, and the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH, primers sets from Sangon Technologies, Shanghai, China) was amplified for normalization. Quantitative PCR data were analyzed using the StepOnePlus™ Real-Time PCR system (Applied Biosystems). The relative RNA levels with different incubation time were calculated by the 2−ΔΔCT method using GAPDH as a control.
Cell viability assay
NCI-H460 or other cells were seeded in 96-well plates at 5 × 103 cells per well overnight. The medium was then removed, and the cells were treated with RNA molecules in fresh medium at different concentrations. After 48 h of incubation, cell viability was determined using Cell Counting Kit-8 (CCK-8; Dojindo, Tokyo, Japan). The absorbance was measured at 450 nm using a microplate reader (Thermo Fisher Scientific, Rockford, IL).
In vivo imaging analysis
After the tumor grew to 200~300 mm3, tumor-bearing mice were administered with Cy5.5-labeled RNA molecules by tail vein injection. Cy5.5-labeled RNA molecules were tracked using an in vivo imaging system (Kodak FX Pro; Carestream Health, Rochester, NY) at 0.5, 2, and 3.5 h post-injection18. Tumors were then exacted for imaging after 4 h of circulation.
In vivo trap assay with qRT-PCR
Three mice were administered with 1 nmol syn-RA16 or trans-RA16 via intravenous injection. After 3.5 h of circulation, tumor, heart, liver, lung, and kidney tissues were collected for RNA extraction using TRIzol reagent (Thermo Fisher Scientific, Rockford, IL) according to the manufacturer’s protocol. The resulting total RNA was first treated with DNase I to eliminate DNA contamination and quantified using One Drop (Hong Kong, China). Next, 500 ng of DNase I-treated RNA was reverse-transcribed into DNA using M-MLV transcriptase (TaKaRa, Dalian, China). qPCR was performed with RA16 aptamer primers and Power SYBR Green Master Mix (Life Technologies) according to the manufacturer’s protocol, and mouse 18S RNA (primers sets from Sangon Technologies, Shanghai, China) was amplified for normalization18. qPCR data were analyzed using the StepOnePlus™ Real-Time PCR system (Applied Biosystems). The relative RNA levels in various tissues were calculated by the 2−ΔΔCT method using mouse 18S RNA as the control46,47.
Truncation of RA16 aptamers
RA16 aptamers were truncated into three parts.
S1: 5′-GGGUGCCAAGCCGUCGGGUUAUGUUGAUCUCCUCAAGGAC-3′. S2: 5′-GGGUGCCAAGCCGUCGGGUUAUGUUGAUCUCCUCAAGGACGAGUGCAUUGCAUCACGUCAGUAG-3′ S3: 5′-GGGAGAGAACAAUGACCUGCGGUGCCAAGCCGUCGGGUUAUGUUGAUCUCCUCAAGGACGAGUGCAUUG-3′.
S1 was transcribed from the DNA sequence amplified by PCR using 5′-CACTAATACGACTCACTATAGGGTGCCAAGCCGTCGGGTTATGTTGATCTCCTCAAGGACGAGTGCATTGCATCACGTCAGTAG-3′ as template and the underlined sequences as primers.
S2 was transcribed from the DNA sequence amplified by PCR using 5′-CACTAATACGACTCACTATAGGGTGCCAAGCCGTCGGGTTATGTTGATCTCCTCAAGGACGAGTGCATTGCATCACGTCAGTAG-3′ as template and the underlined sequences as primers.
S3 was transcribed from the DNA sequence amplified by PCR using 5′-CACTAATACGACTCACTATAGGGAGAGAACAATGACCTGCGGTGCCAAGCCGTCGGGTTATGTTGATCTCCACAAGGACGAGTGCATTGCATCACGTCAGTAG-3′ as template and the underlined sequences as primers.
The in vitro transcription process was similar to that described for the full-length aptamer with the incorporation of 16-biotin-UTP. The secondary structures of the truncated aptamers, which include the possible binding region, were predicted by Mfold software (http://unafold.rna.albany.edu/?q=mfold).
Statistical analysis
Results are presented as the mean ± standard deviation of at least three independent experiments with duplicate samples. Statistical differences were evaluated using one-way analysis of variance unless otherwise indicated. P < 0.05 was considered as statistically significant. Graphs were generated by GraphPad Prism (version 6; GraphPad, La Jolla, CA, USA) and Microsoft Excel (version 2010).
References
Chen, W. et al. Cancer statistics in China, 2015. CA Cancer J Clin 66(2), 115–132 (2016).
Visbal, A. L., Leighl, N. B., Feld, R. & Shepherd, F. A. Adjuvant Chemotherapy for Early-Stage Non-small Cell Lung Cancer. Chest 128, 2933–2943, https://doi.org/10.1378/chest.128.4.2933 (2005).
Group, N. M.-A. C. Chemotherapy in addition to supportive care improves survival in advanced non-small-cell lung cancer: a systematic review and meta-analysis of individual patient data from 16 randomized controlled trials. J Clin Oncol 26, 4617–4625, https://doi.org/10.1200/JCO.2008.17.7162 (2008).
Bray, F. et al. Global Cancer Statistics 2018: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin 68, 394–424, https://doi.org/10.3322/caac.21492 (2018).
Sabari, J. K. et al. Changing the Therapeutic Landscape in Non-small Cell Lung Cancers: the Evolution of Comprehensive Molecular Profiling Improves Access to Therapy. Curr Oncol Rep 19, 24, https://doi.org/10.1007/s11912-017-0587-4 (2017).
Ruiz-Ceja, K. A. & Chirino, Y. I. Current FDA-approved treatments for non-small cell lung cancer and potential biomarkers for its detection. Biomed Pharmacother 90, 24–37, https://doi.org/10.1016/j.biopha.2017.03.018 (2017).
Raben, D. et al. The effects of cetuximab alone and in combination with radiation and/or chemotherapy in lung cancer. Clin Cancer Res 11, 795–805 (2005).
Thienelt, C. D. et al. Multicenter phase I/II study of cetuximab with paclitaxel and carboplatin in untreated patients with stage IV non-small-cell lung cancer. J Clin Oncol 23, 8786–8793, https://doi.org/10.1200/JCO.2005.03.1997 (2005).
Posey, A. D. Jr. et al. Engineered CAR T Cells Targeting the Cancer-Associated Tn-Glycoform of the Membrane Mucin MUC1 Control Adenocarcinoma. Immunity 44, 1444–1454, https://doi.org/10.1016/j.immuni.2016.05.014 (2016).
Johnson, D. H. et al. Randomized phase II trial comparing bevacizumab plus carboplatin and paclitaxel with carboplatin and paclitaxel alone in previously untreated locally advanced or metastatic non-small-cell lung cancer. J Clin Oncol 22, 2184–2191, https://doi.org/10.1200/JCO.2004.11.022 (2004).
Geyer, M. B. & Brentjens, R. J. Review: Current clinical applications of chimeric antigen receptor (CAR) modified T cells. Cytotherapy 18, 1393–1409, https://doi.org/10.1016/j.jcyt.2016.07.003 (2016).
Shahid, M., Nimjee, R. R. W. & Richard, C. Becker, a. B. A. S. Aptamers as therapeutics. The Annual Review of Pharmacology and Toxicology 57, 61–79, https://doi.org/10.1146/annurev-pharmtox-010716-104558 (2017).
Tuerk, C. & Gold, L. Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 249, 505–510 (1990).
Ellington, A. D. & Szostak, J. W. In vitro selection of RNA molecules that bind specific ligands. Nature 346, 818–822, https://doi.org/10.1038/346818a0 (1990).
Ng, E. W. et al. Pegaptanib, a targeted anti-VEGF aptamer for ocular vascular disease. Nat Rev Drug Discov 5, 123–132, https://doi.org/10.1038/nrd1955 (2006).
Sridharan, K. & Gogtay, N. J. Therapeutic nucleic acids: current clinical status. Br J Clin Pharmacol 82, 659–672, https://doi.org/10.1111/bcp.12987 (2016).
Bunka, D. H. & Stockley, P. G. Aptamers come of age - at last. Nat Rev Microbiol 4, 588–596, https://doi.org/10.1038/nrmicro1458 (2006).
Wang, H. et al. In Vivo SELEX of an Inhibitory NSCLC-Specific RNA Aptamer from PEGylated RNA Library. Molecular Therapy-Nucleic Acids 10, 187–198 (2018).
Li, N., Nguyen, H., Byrom, M. & Ellington, A. D. Inhibition of Cell Proliferation by an Anti-EGFR Aptamer. Plos One 6, e20299, https://doi.org/10.1371/journal.pone.0020299 (2011).
Zhou, J., Li, H., Zhang, J., Piotr, S. & Rossi, J. Development of cell-type specific anti-HIV gp120 aptamers for siRNA delivery. J Vis Exp, https://doi.org/10.3791/2954 (2011).
Bouchard, P. R., Hutabarat, R. M. & Thompson, K. M. Discovery and development of therapeutic aptamers. Annu Rev Pharmacol Toxicol 50, 237–257, https://doi.org/10.1146/annurev.pharmtox.010909.105547 (2010).
Zuker, M. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 31, 3406–3415 (2003).
Liu, T. C., Jin, X., Wang, Y. & Wang, K. Role of epidermal growth factor receptor in lung cancer and targeted therapies. Am J Cancer Res 7, 187–202 (2017).
Thakur, M. K. & Wozniak, A. J. Spotlight on necitumumab in the treatment of non-small-cell lung carcinoma. Lung Cancer (Auckl) 8, 13–19, https://doi.org/10.2147/LCTT.S104207 (2017).
Mayer, G. The chemical biology of aptamers. Angew Chem Int Ed Engl 48, 2672–2689, https://doi.org/10.1002/anie.200804643 (2009).
Vater, A. & Klussmann, S. Toward third-generation aptamers: Spiegelmers and their therapeutic prospects. Curr Opin Drug Discov Devel 6, 253–261 (2003).
Sundaram, P., Kurniawan, H., Byrne, M. E. & Wower, J. Therapeutic RNA aptamers in clinical trials. Eur J Pharm Sci 48, 259–271, https://doi.org/10.1016/j.ejps.2012.10.014 (2013).
Charoenphol, P. & Bermudez, H. Aptamer-targeted DNA nanostructures for therapeutic delivery. Mol Pharm 11, 1721–1725, https://doi.org/10.1021/mp500047b (2014).
Zeng, Z. et al. Specific and sensitive tumor imaging using biostable oligonucleotide aptamer probes. Theranostics 4, 945–952, https://doi.org/10.7150/thno.9246 (2014).
Shi, H. et al. In vivo fluorescence imaging of tumors using molecular aptamers generated by cell-SELEX. Chem Asian J 5, 2209–2213, https://doi.org/10.1002/asia.201000242 (2010).
Ferreira, C. S. M. et al. DNA aptamers against the MUC1 tumour marker: design of aptamer-antibody sandwich ELISA for the early diagnosis of epithelial tumours. Anal Bioanal Chem 390, 1039–1050, https://doi.org/10.1007/s00216-007-1470-1 (2008).
Chen, L. et al. The isolation of an RNA aptamer targeting to p53 protein with single amino acid mutation. P Natl Acad Sci USA 112, 10002–10007, https://doi.org/10.1073/pnas.1502159112 (2015).
Hu, Y. et al. Novel MUC1 aptamer selectively delivers cytotoxic agent to cancer cells in vitro. Plos One 7, e31970, https://doi.org/10.1371/journal.pone.0031970 (2012).
Perepelyuk, M., Sacko, K., Thangavel, K. & Shoyele, S. A. Evaluation of MUC1-Aptamer Functionalized Hybrid Nanoparticles for Targeted Delivery of miRNA-29b to Nonsmall Cell Lung Cancer. Mol Pharm 15, 985–993, https://doi.org/10.1021/acs.molpharmaceut.7b00900 (2018).
Zeller, G. et al. Potential of fecal microbiota for early-stage detection of colorectal cancer. Mol Syst Biol 10, 766, https://doi.org/10.15252/msb.20145645 (2014).
Bates, P. J., Laber, D. A., Miller, D. M., Thomas, S. D. & Trent, J. O. Discovery and development of the G-rich oligonucleotide AS1411 as a novel treatment for cancer. Exp Mol Pathol 86, 151–164, https://doi.org/10.1016/j.yexmp.2009.01.004 (2009).
Shi, H. et al. Whole cell-SELEX aptamers for highly specific fluorescence molecular imaging of carcinomas in vivo. Plos One 8, e70476, https://doi.org/10.1371/journal.pone.0070476 (2013).
Liang, C. et al. Aptamer-functionalized lipid nanoparticles targeting osteoblasts as a novel RNA interference-based bone anabolic strategy. Nat Med 21, 288–294, https://doi.org/10.1038/nm.3791 (2015).
Tan, L. H., Neoh, K. G., Kang, E. T., Choe, W. S. & Su, X. D. PEGylated Anti-MUC1 Aptamer-Doxorubicin Complex for Targeted Drug Delivery to MCF7 Breast Cancer Cells. Macromol Biosci 11, 1331–1335, https://doi.org/10.1002/mabi.201100173 (2011).
Xue, L., Maihle, N. J., Yu, X., Tang, S. C. & Liu, H. Y. Synergistic Targeting HER2 and EGFR with Bivalent Aptamer-siRNA Chimera Efficiently Inhibits HER2-Positive Tumor Growth. Mol Pharm 15, 4801–4813, https://doi.org/10.1021/acs.molpharmaceut.8b00388 (2018).
Hu, L. et al. Selection, Characterization and Interaction Studies of a DNA Aptamer for the Detection of Bifidobacterium bifidum. Int J Mol Sci 18, https://doi.org/10.3390/ijms18050883 (2017).
Rockey, W. M. et al. Rational truncation of an RNA aptamer to prostate-specific membrane antigen using computational structural modeling. Nucleic Acid Ther 21, 299–314, https://doi.org/10.1089/nat.2011.0313 (2011).
Mi, J. et al. In vivo selection of tumor-targeting RNA motifs. Nat Chem Biol 6, 22–24, https://doi.org/10.1038/nchembio.277 (2010).
Shangguan, D. et al. Identification of liver cancer-specific aptamers using whole live cells. Anal Chem 80, 721–728, https://doi.org/10.1021/ac701962v (2008).
Shangguan, D. et al. Aptamers evolved from live cells as effective molecular probes for cancer study. Proc Natl Acad Sci USA 103, 11838–11843, https://doi.org/10.1073/pnas.0602615103 (2006).
Livak, K. J. & Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25, 402–408, https://doi.org/10.1006/meth.2001.1262 (2001).
Cheng, C., Chen, Y. H., Lennox, K. A., Behlke, M. A. & Davidson, B. L. In vivo SELEX for Identification of Brain-penetrating Aptamers. Mol Ther Nucleic Acids 2, e67, https://doi.org/10.1038/mtna.2012.59 (2013).
Acknowledgements
This study was supported by funds from the State New Drug Research & Development (2011ZX09401-027), China Scholarship Council Foundation (201506210382) and the National Postdoctoral Program for Innovative Talent (BX20180210). We are also grateful to Soochow University for animal administration and the technical support provided for in vivo imaging.
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H.W., R.L., X.D., I.C. and Y.J. designed the experiments. H.W. conducted the experiments. H.W. and M.Q. performed data analysis and interpretation. I.C., X.D. and Y.J. provided critical materials and insights. H.W. and Y.J. wrote the manuscript.
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H.W. and M.Q. are employees of Biopharmagen Corp. The other authors declare no potential conflicts of interest.
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Wang, H., Qin, M., Liu, R. et al. Characterization of A Bifunctional Synthetic RNA Aptamer and A Truncated Form for Ability to Inhibit Growth of Non-Small Cell Lung Cancer. Sci Rep 9, 18836 (2019). https://doi.org/10.1038/s41598-019-55280-x
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DOI: https://doi.org/10.1038/s41598-019-55280-x
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