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Self-assembled Tetrahedral DNA Nanostructures Promote Mesenchymal Stem Cell Migration via lncRNA XLOC 010623 and RHOA/ROCK2 Pathway Si-Rong Shi, Qiang Peng, Xiao-Ru Shao, Jing Xie, Shiyu Lin, Tao Zhang, Qianshun Li, Xiaolong Li, and Yunfeng Lin ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b06528 • Publication Date (Web): 12 Jul 2016 Downloaded from http://pubs.acs.org on July 16, 2016
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Self-assembled Tetrahedral DNA Nanostructures Promote Adipose-derived Stem Cell Migration via lncRNA XLOC 010623 and RHOA/ROCK2 Signal Pathway Sirong Shi1, Qiang Peng1, Xiaoru Shao1, Jing Xie1, Shiyu Lin1, Tao Zhang 1, Qianshun Li1, Xiaolong Li1 and Yunfeng Lin1*
1
State Key Laboratory of Oral Diseases, West China Hospital of
Stomatology, Sichuan University, Chengdu 610041, P. R. CHINA.
*Corresponding author: Yunfeng Lin State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu 610041, P. R. CHINA; Tel: +86-28-85503487; Fax: +86-28-85582167; E-mail address:
[email protected]
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ABSTRACT Self-assembled tetrahedral DNA nanostructures (TDNs) with precise sizes have been extensively applied in various fields owing to their exceptional mechanical rigidity, structural stability, and modification versatility. In addition, TDNs can be internalized by mammalian cells and remain mainly intact within the cytoplasm by escaping degradation by nucleases. Here, we studied the effects of TDNs on cell migration and the underlying molecular mechanisms. TDNs remarkably enhanced the migration of rat adipose-derived stem cells and down-regulated the long non-coding RNA (lncRNA) XLOC 010623 to activate the mRNA expression of Tiam1 and Rac1. Furthermore, TDNs highly up-regulated the mRNA and protein expression of Rhoa, Rock2 and Vcl. These results indicate that TDNs suppressed the transcription of lncRNA XLOC 010623, and activated the TIAM1/RAC1 and RHOA/ROCK2 signaling pathways to promote cell migration. On the basis of these findings, TDNs show a high potential for application in tissue repair and regenerative medicine as a functional three-dimensional DNA nanomaterial.
KEYWORDS: self-assembled, TDNs, mechanical rigidity, structural stability, ASCs, cell migration
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1. INTRODUCTION With the emergence of DNA nanotechnology, DNA has been studied, and used as a material for therapeutics and diagnostics for many years.1-5 Recently, increasing attention has been paid to DNA nanostructures, which possess a high resistance to enzymatic degradation in biological media and are an attractive candidate material for applications in biology and biomedicine.6-9 Among the different forms of DNA nanostructures, tetrahedral DNA nanostructures (TDNs) have been identified as one of the most promising types.10-11 TDNs can be simply, rapidly, and reliably self-assembled from ssDNA (single-stranded DNA) at a high yield (≈ 90%). Besides, as a three-dimensional (3D) DNA nanostructure, TDNs have excellent mechanical rigidity, structural stability, and modification versatility.12-15 Previous studies have shown that TDNs can be internalized by mammalian cells even in the absence of a transfection reagent and remain mainly intact within the cytoplasm.6 Meanwhile, it has been shown that TDNs can be used for the intracellular delivery of other biomolecules, such as oligonucleotides.7 Fan et al. demonstrated that TDNs readily enter cells mainly through a caveolin-dependent endocytosis pathway and are then transported to the lysosomes in a microtubule-mediated manner. Furthermore, they designed modified TDNs that would escape from the lysosomes and enter the cell nuclei, by functionalizing the TDNs with nuclear location signals.16 Most significantly, it has been reported that TDNs could efficiently be modified with siRNAs for targeted in vivo delivery.17 These developments create new opportunities for the design of targeted therapies. However, few studies have examined the effects of TDNs on the
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biological activities of cells, and especially little is known about their effects on cell migration. Cell migration is a remarkably complex cellular behavior that is a key component of many biological processes, including embryonic development, tissue regeneration, and immune responses, and it is also an important pathological factor in diseases such as chronic inflammatory disease and cancer metastasis.18-21 For example, after tissue injury, cells migrate towards the wound, accelerate cell division near the edge of the wound, and secrete growth factors to support further cell migration.22-23 Studies on cell motility have been carried out on substrates with a biomimetic nanostructured topology characterized by rough surfaces, and such material have potential applications in supporting wound healing and/or regenerative medicine.24-27 Likewise, cell migration can be spatially and temporally controlled via nanoporous surfaces.28 In mechanistic terms, it has been extensively demonstrated that the reciprocal regulation of the proteins RAC and RHO is crucial for cell migration. RAC appears to be responsible for regulating actin polymerization and membrane protrusion at the leading edge of the cell, while RHO seems to regulate contraction and retraction forces in the cell body and the rear of the cell.29 RHOA kinase (ROCK) is
thought
to
regulate
actin
filament
formation
to
control
cytoskeletal
tension-mediated changes in cell morphology. During cell migration, ROCK may assist the cell contraction process and regulate transmigration through other cell layers.30-31 In addition, RHOA and ROCK control actin–myosin contractility and the retraction of the rear of the cell, thus driving translocation of the cell body.32 However,
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although the regulation of cell migration is understood to some extent, the effect of TDNs on cell migration and the underlying molecular mechanisms remain to be elucidated. Therefore, in this study, we aimed to determine the effects of TDNs on the migration of adipose-derived stem cells (ASCs) and explore the underlying mechanisms. The findings suggested that the RHOA/ROCK2 signaling pathway plays a significant role in mediating the effects of TDNs on the migration of ASCs.
2. MATERIALS AND METHODS 2.1. Materials. Single-stranded DNA (ssDNA) (Table 1) strands with sequences of our design were synthesized by TaKaRa (Otsu, Japan). Fetal bovine serum (FBS), Phosphate-buffered saline (PBS) and minimum essential medium, α modification (α-MEM) were obtained from GE Healthcare (Little Chalfont, UK). Penicillin-streptomycin solution and 0.25% (w/v) trypsin-ethylene diamine tetraacetic acid solution were obtained from Life Technologies (Carlsbad, CA). Type I collagenase was purchased from Biosharp (China). Bicinchoninic acid, Sodium dodecyl sulfate (SDS) sample buffer, Tris-HCl and MgCl2 were purchased from Bio-Rad (Hercules, CA). Polycarbonate Transwell® inserts (8-µm pore diameter), Culture flasks (25-cm2 surface area) and 12-well culture plates were purchased from Corning (New York, NY). Cell invasion /migration plates, (which feature a porous membrane were obtained from ACEA (California, USA)). Polyvinylidene fluoride membrane was purchased from Millipore (Massachusetts, USA). All antibodies were
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purchased from Abcam (Cambridge, UK). FITC-labeled phalloidine and 4ʹ, 6-diamidino-2-phenylindole (DAPI) were purchased from Sigma (St Louis, Mo, USA). Paraformaldehyde solution (4% w/v) was obtained from Boster (Wuhan, China). RNeasy® Plus Mini Kit was bought from Qiagen (Hilden, Germany). DNase I was obtained from Fermentas (Burlington, Canada) and SYBR® Green I polymerase chain reaction (PCR) master mix was purchased from TaKaRa (Tokyo, Japan).
2.2. Cell Cultures. Animal materials used for this study were obtained according to governing ethical principles and our protocol was reviewed and approved by our Institutional Review Board of The University of Sichuan. ASCs were obtained from subcutaneous adipose tissue of 5-day old Sprague–Dawley female rats. Briefly, collected adipose tissue was cut into small pieces and digested with 0.75% type I collagenase at 37 °C with vigorous agitation for 30min. Enzyme activity was neutralized by the 1:1 (v/v) addition of fresh α-MEM containing 10% (v/v) FBS and 1% (v/v) penicillin-streptomycin solution. The mixed suspension was centrifuged at 200 x g for 5min. After removing the first supernatant, α-MEM containing 10% FBS was added to the centrifuge tubes to re-suspend the ASCs. Subsequently, the ASCs in suspension were seeded into culture flasks (25 cm2 surface area) and incubated at 37 °C under a humidified atmosphere of 5% CO2 until use. Purified ASCs could then be obtained after two passages as described previously.33-35
2.3. Preparation of Tetrahedral DNA Nanostructures. Tetrahedral DNA nanostructures were prepared as reported previously.36 In brief, four single-stranded DNA strands of our design were synthesized by TaKaRa Bio and
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mixed in an equimolar ratio in TM buffer (10mM Tris-HCl, pH 8.0, 50mM MgCl2). The solution was heated to 95 °C for 10min and then quickly cooled to 4 °C for 20min.
2.4. Atomic Force Microscopy (AFM) Analysis. TDNs were diluted to 20nM in TM buffer supplemented with ddH2O in a 200-µl volume. Ten microliters of the diluted TDN solution was dripped onto freshly cleaved mica and dried for 15min. AFM measurements were performed in tapping mode using a SPM-9700 instrument (Shimadzu, Kyoto, Japan).
2.5. Parallel Cell Migration (Wound Healing Assay). The migration of ASCs cultured under different conditions was determined by bidirectional wound healing assay. Briefly, cells were seeded in 12-well plates (1 × 105 cells/well), incubated at 37 °C and 5% CO2 with 10% FBS for 24h, and then grown to 80–90% confluence in 12-well plates, after which a sterile pipette tip was used to scratch the monolayer of cells to form a bidirectional wound. Cell debris was washed away with PBS. Cells were exposed to medium containing 1% FBS only (control) and the same medium containing TDNs (concentration from 62.5–250nM). Wound closure was photographed 0, 12, and 24h after incubation.
2.6. Vertical Cell Migration (Transwell® Chamber Assay). Vertical ®
cell migration assays were performed using polycarbonate Transwell inserts (8-µm ®
pore diameter). Transwell inserts were placed in a 12-well culture plate containing 200µl of α-MEM media containing 1% FBS. In the upper half of the insert 1 × 105 cells were placed inside the chamber. TDNs were added into the upper chamber at the
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concentration of 250nM. After 24h, the cells in the insert were washed with PBS, fixed with 4% (w/v) paraformaldehyde and stained with DAPI for 15min. the number of cells present in the lower part of each insert was determined by counting cells in five microscopic fields of view per well, and the extent of migration was expressed as the mean number of cells per microscopic field of view.
2.7. Cellular Uptake of TDNs. Cell images were taken with a TC5 SP5 confocal microscope (Leica, Wetzlar, Germany). ASCs were seeded in a confocal culture dish at a density of 1 × 104 cells/ml and incubated at 37 °C for 24h. They were then washed twice with PBS and incubated with ssDNA labeled with Cy3 on strand 1 (250nM) and TDNs fluorescently labeled with Cy3 on strand 1 (250nM) in fresh α-MEM media for 6h at 37 °C, respectively. Cells were then washed three times with PBS and fixed with 4% (w/v) paraformaldehyde. The cytoskeleton and nuclei were stained with FITC-labeled phalloidin and DAPI, respectively.
2.8. Real-time Cell Analysis (RTCA) Migration/Invasion Assays. Real-time cell motility was monitored using an xCELLigence® DP RTCA instrument (Roche Diagnostics, Basel, Switzerland) according to the manufacturer’s standard protocol. Briefly, cells (6 × 105 cells/well) were seeded onto the upper chamber of a cell invasion/migration plate, which features a porous membrane. Migrating cells attach and migrate directly through the pores to the underside of the membrane, where they are detected by electrodes connected to the membrane. Prior to cell seeding, 165µl fresh culture medium containing 1% FBS was added to lower chamber and 30µl of blank medium containing 1% FBS was added to the upper chamber, and then
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the solutions were left to equilibrate in the incubator for 1h at 37 °C and 5% CO2. After this incubation step, a background reading was taken for each well, and then 6 × 105 cells/well suspended in culture medium containing 1% FBS were seeded in the upper chamber according to the manufacturer’s manual (Roche Diagnostics). TDNs were added into the upper chamber at various concentrations (62.5–250 nM). The cell invasion/migration plates were placed onto the RTCA station and the cell indices were measured every 15min for up to 48h with the RTCA software.
2.9. Western Blot Analysis. After treatment with TDNs or vehicle control for 24 h, cells were washed three times with ice-cold PBS, then harvested and lysed in lysis buffer containing protease inhibitors. The lysates were centrifuged at 10310 × g for 5min at 4 °C. The supernatant was collected, and protein concentration was determined by bicinchoninic acid assay. Protein samples were solubilized and boiled in SDS sample buffer for 5min and then separated using SDS-polyacrylamide gel electrophoresis at 100V for 90min on a 10% (v/v) polyacrylamide gel. Subsequently, the separated proteins were transferred to a polyvinylidene fluoride membrane. Following incubation in blocking solution consisting of 5% (w/v) bovine serum albumin in Tris-buffered saline containing 0.05% (v/v) Tween® 20 for 1h at room temperature and overnight incubation at 4 °C with a primary antibody specific to GAPDH, RHOA, ROCK2, or VCL, the membrane was washed and then probed with an appropriate secondary antibody for 1h at room temperature. After washing three times with TBST, hybridization was visualized using enhanced chemiluminescence reagent. The abundance of each protein of interest was compared to that of the loading
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control (GAPDH) based on the relative intensities of the bands.
2.10. Immunofluorescence Staining. Cells were seeded in a confocal microscopy culture dish and incubated at 37 °C and 5% CO2 for 8h. After washing three times with ice-cold PBS, TDNs were added into the dish in fresh α-MEM media and the cells were incubated for a further 24h. After incubation, cells were fixed with 4% (w/v) paraformaldehyde for 30min, permeabilized with 0.1% (v/v) Triton™ X-100 for 15min, blocked by adding 1% (v/v) goat serum in PBS for 1.5h at room temperature, and incubated in primary antibody solution (1:100 dilution, anti-VCL, anti-RHOA, or anti-ROCK2 rabbit polyclonal antibody) at 4 °C overnight. Next, DyLight®594 goat anti-rabbit monoclonal secondary antibodies (1:200 dilutions) were incubated with the cells for 1h at 37 °C. Subsequently, the cytoskeleton and nuclei were stained with FITC-labeled phalloidine for 30min and DAPI for 15min, respectively. Each step was followed by washing with PBS for 5min three times. Finally, the cells were mounted using 10% (v/v) glycerin and imaged by confocal laser scanning microscopy.
2.11. Quantitative Real-time PCR (qPCR). Total RNA was extracted from ASCs using an RNeasy® Plus Mini Kit with a genomic DNA eliminator. Extracted RNA samples were dissolved in RNase-free water and quantified by measuring the absorbance at 260nm with a spectrophotometer. After treatment with DNase I, around 0.5µg of each total RNA sample was reverse transcribed using a first strand cDNA synthesis kit (Fermentas). To evaluate the expression of target mRNAs (Table 2) in each treatment group, qPCR was performed using SYBR® Green I PCR
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master mix and an ABI 7300 thermal cycler (Applied Biosystems, Foster City, CA). The selected sets of primers are listed in Table 2. All primers were designed using BLAST searches and amplification of Actb was used as a control for assessing the efficiency of the PCR experiments. Amplification of each target mRNA was performed by qPCR using the following procedure: Denaturation for 3 min at 94 °C, followed by 40 cycles of 5 s at 94 °C and 34 s at 60 °C. For each reaction, a melting curve was generated to test for primer dimer formation and false priming.
2.12. Statistical Analysis. All experiments were performed in triplicate and reproduced at least three times. Statistical analysis of data was performed using SPSS 19.0 (IBM, Armonk, NY) using one-way analysis of variance to compare the means of all groups, and the Student–Newman–Keuls test to compare the means of each pair of groups. Group means were considered to be significantly different if the two-tailed p value was < 0.05.
3. RESULTS AND DISCUSSION TDNs were readily assembled from four 63-base ssDNA strands (Table 1, Strands1–4) via a simple annealing process using a thermal cycler (Bio-Rad, Hercules, CA).12, 19 Each ssDNA strand forms one of the four faces of the DNA tetrahedron by hybridizing to the other three ssDNA strands running around the adjacent faces at the shared edges (Figure 1A). The TDNs were characterized by AFM, which has previously been used to verify the successful self-assembly of TDNs.7, 37 The results of the AFM experiments showed that the TDNs in the dried state were approximately
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2–3 nm in height (Figure 1B), which is in accordance with previous studies.38-40 Meanwhile, as shown in Figure 1C, high-resolution images identified the 3D structure of TDNs as well as confirming a height of ~3 nm. Taking all these results together, TDNs appeared as well-dispersed, uniformly-sized, triangular particles. Subsequently, to investigate the effects of TDNs on cell migration of ASCs, RTCA was used, which is a technique that uses real-time cell monitoring to detect migration and invasion. RTCA was carried out with an xCELLigence® DP system, which enables continuous data recording over a period of several days. In marked contrast to endpoint assays, RTCA allows for the precise determination of the kinetics of the migratory and invasive activities of a given cell population in culture.41-42 It can be clearly observed from Figure 2A that TDNs markedly promoted the cell migration of ASCs in a concentration-dependent manner from 62.5–250 nM during the 60 h test period. In particular, TDNs at the highest tested concentration of 250 nM had the strongest effect on the promotion of cell migration throughout the test period. Statistical
analysis
of
the
RTCA
results
showed
that
a
significant
concentration-dependent promotion of cell migration by TDNs was detected when the ASCs were treated with TNDs for all tested incubation periods of 12, 24, 36, and 48h (Figure 2B). With the objective of studying the effects of TDNs on the horizontal migration of ASCs, we treated ASCs with different concentrations of TDNs and assayed their migration using an in vitro bidirectional wound healing assay. Wound healing assays have been widely used for detecting the migration of cells.43-44 As shown in Figure 3A,
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cells migrated towards the wound area over time from 0–24h, and the groups incubated with TDNs (62.5, 125, and 250 nM) clearly migrated faster than the control group did. The observations were confirmed by calculating the migration rate of each treatment group, and a statistical analysis (Figure 3B) showed that the migration of ASCs
was
significantly
improved
in
the
presence
of
TNDs
in
a
concentration-dependent manner. Especially, TDNs notably and significantly (***p< 0.001) enhanced the migration of ASCs, and the strongest effect was observed at the highest tested concentration of 250 nM. Combining the results of RTCA with those of the wound healing assay, it can be concluded that the migration of ASCs was remarkably enhanced with an increasing concentration of the TDNs from 62.5–250 nM, and the concentration of 250 nM resulted in the strongest promotion of cell migration. To further explore the effect of TDNs on the migration of ASCs, Transwell® chamber assays were also performed. As illustrated in Figure 4A, the ASCs were seeded into the upper chamber of each Transwell® insert, then the medium was exchanged for fresh α-MEM containing 1% FBS after 8h. Finally, TDNs (250 nM) were added to the upper chamber for 24 h. In comparison to the control group, more cells were found to have migrated to the lower surface of the Transwell® insert in the group treated with 250 nM TDNs (Figure 4B shows representative images). Moreover, quantitative analysis indicated that treatment with TDNs markedly enhanced the migration of ASCs (***p
