Self-Assembled Tetrahedral DNA Nanostructures Promote Neural

Subscriber access provided by READING UNIV

Article

Self-Assembled Tetrahedral DNA Nanostructures Promote Neural Stem Cell Proliferation and Neuronal Differentiation Wenjuan Ma, Xiaoru Shao, Dan Zhao, Qianshun Li, Mengting Liu, Tengfei Zhou, Xueping Xie, Chenchen Mao, Yuxin Zhang, and Yunfeng Lin ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b00833 • Publication Date (Web): 09 Feb 2018 Downloaded from http://pubs.acs.org on February 11, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Applied Materials & Interfaces is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

Self-Assembled Tetrahedral DNA Nanostructures Promote Neural Stem Cell Proliferation and Neuronal Differentiation Wenjuan Ma, Xiaoru Shao, Dan Zhao, Qianshun Li, Mengting Liu, Tengfei Zhou, Xueping Xie, Chenchen Mao, Yuxin Zhang, Yunfeng Lin*

State Key Laboratory of Oral Diseases, National Clinical Research Center for Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu 610041, P. R. China;

*Correspondence Author: Yunfeng Lin State Key Laboratory of Oral Diseases, National Clinical Research Center for Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu 610041, P. R. China; Tel: 86-28-85503487; Fax: 86-28-85503487 Email address: [email protected]

Keywords: tetrahedral DNA nanostructure, NE-4C stem cells, proliferation, differentiation, Notch signaling pathway

1

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract Stem cell-based therapy is considered a promising approach for repair of nervous tissues. Neural stem cells (NSCs) cannot proliferate or differentiate efficiently; hence, different biomaterials have been explored to improve NSCs proliferation and differentiation. However, these agents either had low bioavailability or poor biocompatibility. In this work, our group investigated the effects of tetrahedral DNA nanostructures (TDNs), a novel DNA biological material, on the self-renew and differentiation of neuroectodermal (NE-4C) stem cells. We observed that TDN treatment promoted the self-renew of the stem cells via activating the Wnt/β-catenin pathway. In addition, our findings suggested that NE-4C stem cells neuronal differentiation could be promoted effectively by TDNs via inhibiting the Notch signaling pathway. In summary, this is the first report about the effects of TDNs on the proliferation and differentiation of NE-4C stem cells and the results demonstrate that TDNs have great potential in nerve tissue regeneration.

2


Page 2 of 29

Page 3 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1. INTRODUCTION Despite intensive research for decades, therapeutics for diseases of the nervous system, such as neurodegenerative diseases, nerve injuries caused by trauma and hypoxic-ischemic brain injury, remain poorly developed.

1-6

Because it is difficult for nerve cells to self-renew

and self-heal spontaneously, therapies based on the transplantation of neural stem cells (NSCs) have recently gained increasing attentions, and this type of therapy has become a research focus in biomedical fields. 7-15 While much work remains to be done before achieving clinical application of stem cell therapy for neurological diseases, a major challenge that needs to be addressed urgently is that transplanted NSCs cannot proliferate or differentiate effectively.

16, 17

In previous work,

researchers explored the capability of biomolecules such as prostaglandin E2 and Tenuigenin in promoting NSCs proliferation and differentiation in vitro. 18-23 However, these agents have poor performance with regard to either biocompatibility or bioavailability, which are is both necessary for them intended to be used in biomedicine. Therefore, novel biomaterials are urgently needed. In this regard, tetrahedral DNA nanostructures (TDNs), DNA-based nano-biomaterials, have been investigated extensively for their ability to promote cells proliferation and differentiation. It is widely known that DNA nanotechnology have been used in different biomedical applications, such as reversal of tumor multi-drug resistance, targeted drug delivery, and diagnosis of diseases.

24-38

synthesis and high yields.

TDNs have many advantages, including ease of use, ease of

31

The most important advantage of TDNs is their high biological

safety attributable to the biological nature of DNA, which could be degraded by cells. 29 3



Page 4 of 29

Self-assembled TDN comprises four single-stranded DNAs (ssDNAs), and its design is based on complementary base pairing. In contrast to natural DNA, which could be taken up few by cells without the aid of other auxiliary agents, TDNs could be internalized largely by cells via caveolin-mediated endocytosis.

29, 31-33

Some previous literatures have suggested that TDNs

could exert effects on changing the biological behaviors of cells, such as the maintaining rat chondrocyte phenotype, promoting the proliferation of mouse L929 fibroblasts and migrating rat adipose-derived stem cells.

27, 31-33, 39

However, little is known about how TDNs alter the

behavior of undifferentiated and differentiating NSCs, and whether the underlying mechanisms are associated with changes in signaling pathways. To investigate the changes that occur in NSCs upon exposure to TDNs, in this work, we tested the effects of TDNs on neuroectodermal (NE-4C) stem cells self-renew and differentiation. NE-4C stem cells have the ability to proliferate and differentiate into the neuronal lineage and are one of the in vitro models of the nervous system.22,

23

We

hypothesized that TDNs could promote the self-renew and differentiation of NE-4C stem cells and could be applied for neural tissue regeneration and repair. 2. MATERIALS AND METHODS 2.1. Materials The four ssDNAs (Table 1) with specific sequence was synthesized and purified by TaKaRa (Dalian, China). Fetal bovine serum (FBS) and 0.25% (w/v) trypsin-EDTA were obtained from Corning (New York, USA). Dulbecco’s modified Eagle’s medium (DMEM), Dulbecco’s

modified

Eagle’s

(DEM)/F-12

(1:1)

medium

(DMEM/F-12),

and

penicillin-streptomycin solution were purchased from Gibco (Grand Island, NY). Culture 4


Page 5 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


flasks (25 cm2 surface area), culture plates and confocal dishes were acquired from Corning (New York, USA). Tris-HCl and MgCl2 were purchased from Bio-Rad (Hercules, CA). 4’6-diamidino-2-phenylindole (DAPI), FITC-labeled phalloidine and B-27 were obtained from Sigma (St Louis, MO). Paraformaldehyde solution (4% w/v) was purchased from Boster (Wuhan, China). Cell counting kit-8 (CCK-8) was obtained from Shanghai Dojindo Technology Chemical Corp. (Shanghai, China), and DNA Content Quantitation Assay (Cell Cycle) and Whole Cell Lysis Assay were bougt from KeyGEN Institute of Biotechnology (Jiangsu, China). RNeasy Plus Mini Kit and genomic DNA eliminator were obtained from Qiagen (Hiden, Germany). Polyvinylidene fluoride membranes, PrimeScript RT-PCR Kit and cDNA synthesis Kit were obtained from TaKaRa (Dalian, China). All antibodies were obtained from Abcam (Cambridge, U.K). 2.2. Cell Culture Mouse NE-4C stem cells (CRL-2925, ATCC, VA) were cultured with DMEM containing 10% (v/v) FBS and 1% (v/v) Penicillin-streptomycin solution. The growth medium was changed 2 times per week. The cells were placed in a humidified incubator at 37°C and 5% CO2. Only when explored the differentiation of stem cells, were the cells cultured with DMEM/F-12 containing 1% (v/v) B-27 and 1% (v/v) Penicillin-streptomycin solution. 2.3. Synthesis of TDNs According to Table 1, TDN was composed of four different ssDNAs (denoted as S1, S2, S3, and S4) and synthesized by a previously reported method.

31-33, 40, 41

Equal concentrations of

four different ssDNAs were added to TM buffer (10 mM Tris-HCl, 50 mM MgCl2, pH = 8.0) and the solution was mixed. The mixture was heated to 95°C for 10 min and then cooled to 5



4°C for 30 min rapidly. 2.4. Characterization of TDNs To confirm the successful synthesis of TDNs, we measured the molecular weight of the TDNs and the four ssDNAs using 8% PAGE. To further ensure the successful synthesis of TDNs, transmission electron microscopy (TEM, HITICHI, HT7700, Japan) was performed to observe the morphology of TDNs.

42

A drop of TDNs was placed on the specimen and dried

under infrared ray for 5-10 min, and then the samples were imaged by TEM. 2.5. Uptake of TDNs by Cells To prove that TDNs exert effects on NE-4C stem cells, TDNs (Cy5-TDNs, 250 nM) and ssDNA (Cy5-ssDNA, 250 nM) assembled with Cyanine-5 (Cy5) were used to treat cells. First, the NE-4C stem cells were plated onto confocal dishes and cultured in growth medium for 24 h. Then, the medium was replaced with medium without FBS, and 250 nM Cy5-TDNs or 250 nM Cy5-ssDNA were added to the cells for 12 h. All cell samples were washed with PBS thrice and then fixed in cold 4% paraformaldehyde solution for 15 min. After the samples were rinsed again thrice, phalloidine and DAPI were used to stain the cytoskeleton and nucleus, respectively, for 10 min. The samples were rewashed with PBS for three times. Finally, the samples were imaged using a confocal laser microscope (TCS SP8; Lesic, Wetzlar, Germany). 2.6. Proliferation Assay CCK-8 and Cell Cycle Assay were used to analyze cell proliferation. 43 For CCK-8, NE-4C stem cells were seeded into a 96-well plate at a density of 5000 cells per well, and cultured in growth medium for 24h. Then, the growth medium was replaced with medium without FBS 6


Page 6 of 29

Page 7 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


and containing TDNs at concentrations of 62.5 nM, 125 nM, 250 nM, 300 nM, , and 375 nM. NE-4C stem cells proliferation was tested by CCK-8 solution after 24 h. In order to analyze the cell cycle by flow cytometry, NE-4C stem cells were incubated in growth medium for 24h. The medium was then changed to serum-free medium mixed with 250 nM TDNs and cells were put in an incubator at 37°C and 5% CO2. In control groups, the cells were treated without TDNs. After treatment 24 h, all samples were harvested by 0.25% (w/v) trypsin-EDTA solution, rinsed with PBS thrice, and then fixed in cold 70% ethanol at -20°C overnight. The next day, all samples were incubated in 100 µL of RNase at 37°C for 30 min and then stained with 400 µL of propidium iodide (PI) solution at 4°C for 30 min in the absence of light. Finally, the percentage of cells in the G1, S, and G2 phases was determined with a flow cytometer (FC500 Beckman, IL, USA), and the change in the cell cycle stage distribution was analyzed using the software WinMDI2.9 and Cycle 32. 2.7. Differentiation of NE-4C Stem Cells after TDNs Treatment Stem cells were dissociated using 0.25% (w/v) trypsin-EDTA solution, seeded on 6-well plates, and cultured in growth medium for 1 day before neuronal differentiation induction. The growth medium was changed to serum-free medium (SFM), which contained DMEM/F-12, 1% penicillin-streptomycin, and 1% B-27. Previous studies have demonstrated that SFM could promote and support stem cells differentiation into neurons.

22, 23, 44, 45

To

determine whether TDNs could influence NE-4C stem cells differentiation, we added TDNs to SFM at a concentration of 250 nM. SFM mixed with TDNs was subsequently replaced at the same time every day, and cells were induced to differentiate for 7 days. For the control groups, SFM was used without TDNs for 7 days. Lysates were harvested on days 1, 3, and 7 7



for extraction of total RNA and proteins. 2.8. Quantitative Polymerase Chain Reaction As for undifferentiated stem cells, mRNA levels of β-catenin, Lef-1, and Cyclin-D (sequences of mRNA primers are shown in Table 2) were determined after 24 h. In differentiated cells, the mRNA levels of β-III-tubulin (1, 3 and 7 days), Notch-1 (7 days), Hes-1 (7 days), and Hes-5 (7 days) (sequences of mRNA primers are shown in Table 2) were determined. All RNA was extracted from all cells totally by using an RNeasy Plus Mini Kit and genomic DNA eliminator. A cDNA synthesis kit was used for cDNA preparation, and the final volume of cDNA was 20 µL. A PrimeScript RT-PCR Kit was used to complete the qPCR. Amplification of each mRNA was performed by qPCR using the conditions as following: denaturation at 94°C for 3 min, followed by 40 cycles of 5 s at 94°C and 60 °C for 34 s. To detect primer dimer formation and incorrect priming, a melting curve was generated for each reaction. Amplification of GAPDH was used as a control for analyzing the efficiency of the qPCR experiments. 2.9. Western Blot Undifferentiated NE-4C stem cells were cultured in 6-well plates with growth medium for 24 h, and then exposed to medium containing TDNs (250 nM) or without TDNs for 24 h. In order to induce cells differentiation, stem cells were also plated in 6-well plates with growth medium, but after 1 day, the cells were cultured in SFM containing TDNs (250 nM) for 1, 3, and 7 days. PBS was used to wash all cell samples three times, and Whole Cell Lysis Assay was used to extract total proteins. The 5xloading buffer was mixed with proteins samples in a ratio of 1:4, and homogeneously mixed samples were boiled for 4 min. SDS-PAGE at various 8


Page 8 of 29

Page 9 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


gel concentrations was used to separate the target proteins,

32, 46

which were subsequently

transferred to polyvinylidene fluoride membranes. After incubation in blocking buffer containing 5% skim milk powder for 1 h at 37°C, the membranes were incubated overnight with primary antibodies against β-catenin, Lef-1, Cyclin-D, β-tubulin, Notch-1, Hes-1, or Hes-5 (1:1000) at 4°C. The next day, the membranes were washed with Tris-buffered saline containing Tween (TBST) three times. Next, the membranes were incubated in secondary antibodies (1:3000) at 37°C for 1 h. The membranes were then washed three times with TBST, and each membrane was subjected to an enhanced chemiluminescence detection system (Bio-Rad, Hercules, CA, USA). The internal control was GAPDH because of its stable expression in cells. 2.10. Immunofluorescence Staining To confirm that the NE-4C stem cells were undifferentiated, one of the marker proteins secreted by NSCs, Nestin, was examined by confocal laser microscopy (TCS SP8; Leica, Wetzlar, Germany). 47 First, stem cells were cultured on confocal plates with growth medium. After 24h, the cells were rinsed with PBS for three times, fixed in cold 4% paraformaldehyde solution for 15 min. Next, the cells were exposed to 0.5% Triton X-100 for 10 min, washed with PBS, and blocked with 5% sheep serum at 37°C for 1 h. After three washes with PBS, the cells were incubated overnight with antibody against Nestin (1:500) at 4°C and then incubated with a relevant fluorescent secondary antibody at 37°C for 1 h. Next, the cell samples were treated with phalloidin and DAPI for 10 min to stain the cytoskeleton and nucleus, respectively. Finally, images were captured by a confocal laser microscope (TCS SP8; Leica, Wetzlar, Germany). In order to detect the neuronal marker protein (β-III-tubulin), 9



we stained samples that were differentiated for 3 and 7 days with relevant antibodies. Cells were also plated and cultured with growth medium for 1 day, and the medium was changed to SFM containing TDNs (250 nM), which was changed every day. After 3 or 7 days, the samples were washed three times using PBS and fixed with 4% cold paraformaldehyde solution for 15 min, followed by three additional washes with PBS. Differentiated cells were permeabilized with 0.5% Triton X-100 for 10 min, washed three times with PBS, and blocked with 5% sheep serum at 37°C for 1 h. After washed with PBS, the cells were incubated with primary antibody against β-III-tubulin (1:500) at 4°C for 1 day, and on the next day, samples were incubated with a specific fluorescent secondary antibody for 1 h at 37°C. Next, the samples were treated with DAPI for 10 min to stain the nucleus. Finally, a confocal laser microscope (TCS SP8; Leica, Wetzlar, Germany) was used to capture images. 2.11. Statistical Analysis SPSS 19.0 (IBM, Silicon Valley, CA, USA) was used for Statistical analysis. Student’s t-test and one-way analysis of variance (ANOVA) were applied. *p < 0.05, **p < 0.01, and ***p < 0.001 indicated statistical test results were statistically significant. Quantitative results were presented as mean ± standard deviation (SD) with n ≥ 3 replicates.

3. RESULTS AND DISCUSSION 3.1. Characterization of TDNs According to Figure 1A, each ssDNA could self-assemble to form a triangle and then pair with other ssDNA to form a tetrahedral structure via a highly specific base complementation pairing rule. As shown in Figure 1B, PAGE was also used to confirm the successful synthesis 10


Page 10 of 29

Page 11 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


of TDNs according to previous reports. 27, 32 The result of PAGE was consistent with the theoretical value that a TDN is composed of four different ssDNA molecules.40 To further confirm that the synthesis of TDNs was successful, TEM was applied to observe TDNs morphology shaped like triangle, and the particle size of each TDN is 10 nm approximately (Figure 1C). These results successfully confirmed the self-assembly of TDNs. 3.2. Authentication of Undifferentiated NE-4C Stem Cells Undifferentiated NE-4C stem cell has the ability to proliferate. We therefore authenticated whether NE-4C stem cells were differentiated using immunofluorescence. As shown in Figure 2, all cells in the field of view were Nestin antibody-positive, which indicated that the NE-4C stem cells were undifferentiated. 3.3. Uptake of TDNs by NE-4C Stem Cells As reported before, the rapid uptake of TDNs into cells was possible via caveolin-mediated endocytosis.

29

However, ssDNA could not be taken up by the cells in abundance. 29, 32, 33

Moreover, previous studies have shown that cellular uptake of TDNs played an important role in various functions of TDNs. 27, 32, 33 In order to further confirm that NE-4C stem cells could exhibit intracellular uptake of TDNs in abundance, but not of ssDNA, NE-4C stem cells were exposed to Cy5-loaded TDNs (250 nM) or Cy5-loaded ssDNA (250 nM). Figures 3A and 3B showed that the Cy5 fluorescence signal in cells treated with ssDNA was much weaker than that of TDNs modified with Cy5, indicating that TDNs could be taken up by NE-4C stem cells specifically, fulfilling a prerequisite for TDNs to perform their biological activities. 3.4. TDNs Increase NE-4C Stem Cells Proliferation 11



Page 12 of 29

Previous studies have shown that TDNs could effectively promote the proliferation of rat chondrocytes and mouse L929 fibroblasts. 27, 32 In this study, we tried to assess whether this finding is applicable to NE-4C stem cells by cell counting kit-8 (CCK-8) and flow cytometry. Compared with the group without TDNs treatment, the proliferation of NE-4C stem cells treated with TDNs increased with increasing TDNs concentration, which ranges from 62.5 to 375 nM (Figure 4A). We also found that the optimal TDNs concentration for NE-4C stem cells to self-renew was 250 nM, which is consistent with previous research by our group. 31-33

27,

Therefore, the concentration of TDNs used in subsequent experiments was 250 nM. To further confirm the results of the CCK-8, we applied flow cytometry to analyze the

cell cycle of NE-4C stem cells exposed to TDNs for 24 h (Figures 4C and 4D). The number of cells in the S and G1 phases showed significant increase and decrease, respectively. The change of the cell cycle stage distribution showed significant differences between the control and experimental groups (Figure 4B). In the cell cycle, the S phase plays a vital part in DNA replication. Therefore, we concluded that TDNs modulated cell cycle progression to promote the stem cells proliferation, which was in accordance with the result of CCK-8. 3.5. TDNs Accelerate the Process of NE-4C Stem Cells Differentiation into Neurons We could observe that TDNs could promote NE-4C stem cells proliferation, but whether TDNs could affect neurogenesis was still unclear. To address this question, we stimulated NE-4C stem cells with SFM mixed with TDNs (250 nM), and found that the stem cells could then differentiate. Neuron repairing plays a pivotal part in curing many diseases of the nervous tissue;

9, 10

therefore, if TDNs could promote cells differentiation into neurons is

meaningful for our objective in this study. First, we used western blot and qPCR to determine 12


Page 13 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


the expression of target proteins and genes, respectively. β-III-tubulin is a neuronal marker and is secreted by cells when they differentiate into neurons. Figures 5A–C showed that the protein and gene of β-III-tubulin in cells exposed to TDNs were both over-expressed compared with those in control groups after 1, 3 and 7 days. To further observe the phenomenon of NE-4C stem cells differentiation intuitively, we applied immunofluorescence to explore the β-III-tubulin protein expression level. In cells exposed to TDNs for 3 days, confocal laser microscopy imaging revealed a much stronger β-III-tubulin fluorescent signal than that in cells without TDNs treatment (Figure 5D). Figure 5E showed that TDNs promoted cells to over-express the β-III-tubulin significantly. Meanwhile, cells treated with TDNs for 7 days were also analyzed by immunofluorescence. As shown in Figure 5F, the cells treated with TDNs for 7 days also showed a stronger fluorescent signal than those of untreated cells. As shown in Figure 5G, there was a significant difference between the control and experimental samples. Therefore, we inferred that TDNs promoted neuronal differentiation of NE-4C stem cells via up-regulating the β-III-tubulin expression. Importantly, a definite conclusion could be drawn that TDNs could promote NE-4C stem cells differentiation. 3.6. TDNs Promote NE-4C Stem Cells Proliferation via Regulating the Wnt/β-Catenin Pathway TDNs have been reported as promoting cells proliferation by activating the Wnt/β-catenin pathway.

31, 32

In this work, we observed that TDNs also could promote the

self-renew of NE-4C stem cells. This finding may pave the way for the potential use of TDNs in nervous system regeneration. To elucidate the mechanism through which TDNs promote 13



NE-4C stem cells proliferation, the classical Wnt/β-catenin pathway, which is considered vital for the regulation cell cycle, was investigated. The protein and gene expression of β-catenin, Lef-1, and Cyclin-D were determined by western blot and qPCR, respectively. β-catenin is a significant positive mediator of the classical signaling pathway. β-catenin over-expression and accumulation in NE-4C stem cells leads to its transport into the nucleus, where it interacts with lef-1.

48-50

These biological effects could then regulate the self-renew

of NE-4C stem cells. The result of western blot revealed the over-expression of β-catenin, Lef-1, and Cyclin-D in cells exposed to TDNs (Figure 6A). After further data analysis, the effect of TDNs on these proteins was found to be significant (Figure 6B). Meanwhile, qPCR revealed that the levels of β-catenin, Lef-1, and cyclin-D were more high in the groups with TDNs treatment for 24 h than that in the control groups (Figure 6C), and these genes were up-regulated significantly. Importantly, we could observe that the variations in the expressions of these proteins were consistent with the results of gene expression analysis, which indicated that TDNs promoted NE-4C stem cells proliferation through effectively activating the classical Wnt/β-catenin pathway (Figure 7). 3.7. TDNs Promote NE-4C Stem Cell Differentiation into Neuron by Inhibiting the Notch Pathway We have previously observed that TDNs significantly induced differentiation of NE-4C stem cells. This finding indicated that TDNs mighty be helpful in the regeneration of the nervous system. To further verify the mechanism through which TDNs induced cells differentiation, the Notch pathway, which is considered to be associated with cells differentiation, was investigated.

51-55

The proteins and genes expression of Notch-1, Hes-1, 14


Page 14 of 29

Page 15 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


and Hes-5 were determined by western blot and qPCR, respectively. 51, 54 We chose samples that were either exposed to TDNs for 7 days or were not exposed to TDNs. In the cells exposed to TDNs, Notch-1, Hes-1, and Hes-5 were down-regulated. Notch-1, which is expressed on the cell membrane, is an important mediator of the Notch signaling pathway. 53 When Notch-1 expression decreases in differentiated cells, the signal would be carried into the nucleus, thereby down-regulating Hes-1 and Hes-5. These effects eventually could induce the differentiation of cells. The results in Figure 6D demonstrated that Notch-1, Hes-1, and Hes-5 were expressed at a lower level in cells exposed to TDNs for 7 days. After further data analysis, the influence of TDNs on the expression of these proteins was found to be significant (Figure 6E). Additionally, qPCR revealed that the expression of Notch-1, Hes-1, and Hes-5 was lower in cells treated with TDNs for 7 days (Figure 6F), and these genes were down-regulated significantly. Finally, we observed that the variations in the expression of these proteins were consistent with the results of genes expression, indicating that TDNs promoted NE-4C stem cells differentiate to neurons of via inhibiting the Notch pathway. From Figure 7, we could well understand the mechanism of the Notch pathway. 3.8. Discussion Many degenerative diseases influence the nervous system, such as Alzheimer’s disease and Parkinson’s disease; these diseases are characterized by the irreversible loss of neurons and neurophysiological degeneration.

9

To develop effective clinical therapeutics for these

neurodegenerative diseases, many biomaterials have been composited and examined. However, the poor proliferation ability and inefficient differentiation of NSCs for possible therapeutic candidates remain the biggest challenge for the development of effective 15



biological treatments.

16, 17

In the present study, TDNs exhibited high efficacy in promoting

the proliferation and neuronal differentiation of NE-4C stem cells, thus satisfying a key requirement for application to treat neurodegenerative diseases. Based on these results, we speculate that TDNs could act as a potential aid for nerve tissue regeneration. As some studies reported that TDNs were good drug delivery system, we might use TDNs to delivery some medicine to neurocytes.56, 57 Bedsides，the current studies only investigated the effects of TDNs in vitro, therefore, more studies in vivo are needed to conduct.58, 59 We expect that DNA nanomaterials have immense potential for applications in other biological fields. 4. CONCLUSIONS In conclusion, the findings of this study suggested that TDNs promote NE-4C stem cells proliferation via activating the Wnt/β-catenin pathway and accelerate neuronal differentiation by inhibiting the Notch signaling pathway. Furthermore, the most suitable concentration of TDNs was 250 nM, which is consistent with previous studies.27, 31-33 Based on these results, we speculate that TDNs could act as a potential aid for nerve tissue regeneration.

Acknowledgements We would like to acknowledge the financial support from the National Natural Science Foundation of China (81671031, 814702721) and Sichuan Province Youth Science and Technology Innovation Team (2014TD0001). The authors declare no competing financial interest.

References (1) Barkats, M.; Bilang-Bleuel, A.; Buc-Caron, M. H.; Castel-Barthe, M. N.; Corti, O.; Finiels, F.; Horellou, P.;

16


Page 16 of 29

Page 17 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Revah, F.; Sabate, O.; Mallet, J., Adenovirus in the brain: recent advances of gene therapy for neurodegenerative diseases. Prog. Neurobiol. 1998, 55, 333-41. (2) Busl, K. M.; Greer, D. M., Hypoxic-ischemic brain injury: pathophysiology, neuropathology and mechanisms. NeuroRehabilitation 2010, 26, 5-13. (3) Cardone, M., Prospects for gene therapy in inherited neurodegenerative diseases. Curr. Opin. Neurol. 2007, 20, 151-8. (4) Carter, J. E.; Schuchman, E. H., Gene therapy for neurodegenerative diseases: fact or fiction? Br. J. Psychiatry 2001, 178, 392-4. (5) Li, R.; Liu, Z.; Pan, Y.; Chen, L.; Zhang, Z.; Lu, L., Peripheral nerve injuries treatment: a systematic review. Cell Biochem. Biophys. 2014, 68, 449-54. (6) Sabate, O.; Barkats, M.; Buc-Caron, M. H.; Castel-Barthe, M. N.; Finiels, F.; Horellou, P.; Revah, F.; Mallet, J., Adenovirus for neurodegenerative diseases: in vivo strategies and ex vivo gene therapy using human neural progenitors. Clin. Neurosci. 1995, 3, 317-21. (7) Giusto, E.; Donega, M.; Cossetti, C.; Pluchino, S., Neuro-immune interactions of neural stem cell transplants: from animal disease models to human trials. Exp. Neurol. 2014, 260, 19-32. (8) Hoffman, R. M., Nestin-expressing hair follicle-accessible pluripotent stem cells for nerve and spinal cord repair. Cells Tissues Organs 2014, 200, 42-7. (9) Kim, S. U.; Lee, H. J.; Kim, Y. B., Neural stem cell-based treatment for neurodegenerative diseases. Neuropathology 2013, 33, 491-504. (10) Lang, A. E.; Obeso, J. A., Stem cell therapy for Parkinson's disease. Ann. Neurol. 2012, 71, 283; author reply 284. (11) Li, P.; Tessler, A.; Han, S. S.; Fischer, I.; Rao, M. S.; Selzer, M. E., Fate of immortalized human neuronal progenitor cells transplanted in rat spinal cord. Arch. Neurol. 2005, 62, 223-9. (12) Nabetani, M.; Shintaku, H.; Hamazaki, T., Future perspectives of cell therapy for neonatal hypoxic-ischemic encephalopathy. Pediatr. Res. 2017. (13) Niimi, Y.; Levison, S. W., Pediatric brain repair from endogenous neural stem cells of the subventricular zone. Pediatr. Res. 2017. (14) Wei, Y.; Gong, K.; Zheng, Z.; Liu, L.; Wang, A.; Zhang, L.; Ao, Q.; Gong, Y.; Zhang, X., Schwann-like cell differentiation of rat adipose-derived stem cells by indirect co-culture with Schwann cells in vitro. Cell Prolif. 2010, 43, 606-16. (15) Yue, Y.; Yang, X.; Zhang, L.; Xiao, X.; Nabar, N. R.; Lin, Y.; Hao, L.; Zhang, D.; Huo, J.; Li, J.; Cai, X.; Wang, M., Low-intensity pulsed ultrasound upregulates pro-myelination indicators of Schwann cells enhanced by co-culture with adipose-derived stem cells. Cell Prolif. 2016, 49, 720-728. (16) Yang, X. T.; Bi, Y. Y.; Chen, E. T.; Feng, D. F., Overexpression of Wnt3a facilitates the proliferation and neural differentiation of neural stem cells in vitro and after transplantation into an injured rat retina. J. Neurosci. Res. 2014, 92, 148-61. (17) Zhou, X. M.; Sun, J. B.; Yuan, H. P.; Wu, D. L.; Zhou, X. R.; Sun, D. W.; Li, H. Y.; Shao, Z. B.; Zhang, Z. R., A rat model for studying neural stem cell transplantation. Acta Pharmacol. Sin. 2009, 30, 1496-504. (18) Chen, Y.; Huang, X.; Chen, W.; Wang, N.; Li, L., Tenuigenin promotes proliferation and differentiation of hippocampal neural stem cells. Neurochem. Res. 2012, 37, 771-7. (19) Liang, Z.; Shi, F.; Wang, Y.; Lu, L.; Zhang, Z.; Wang, X.; Wang, X., Neuroprotective effects of tenuigenin in a SH-SY5Y cell model with 6-OHDA-induced injury. Neurosci. Lett. 2011, 497, 104-9. (20) Lim, S. T.; Airavaara, M.; Harvey, B. K., Viral vectors for neurotrophic factor delivery: a gene therapy approach for neurodegenerative diseases of the CNS. Pharmacol. Res. 2010, 61, 14-26.

17



(21) Shirosaki, Y.; Hayakawa, S.; Osaka, A.; Lopes, M. A.; Santos, J. D.; Geuna, S.; Mauricio, A. C., Challenges for nerve repair using chitosan-siloxane hybrid porous scaffolds. Biomed Res Int 2014, 2014, 153808. (22) Wong, C. T.; Ahmad, E.; Li, H.; Crawford, D. A., Prostaglandin E2 alters Wnt-dependent migration and proliferation in neuroectodermal stem cells: implications for autism spectrum disorders. Cell Commun Signal 2014, 12, 19. (23) Wong, C. T.; Ussyshkin, N.; Ahmad, E.; Rai-Bhogal, R.; Li, H.; Crawford, D. A., Prostaglandin E2 promotes neural proliferation and differentiation and regulates Wnt target gene expression. J. Neurosci. Res. 2016, 94, 759-75. (24) Dong, S.; Zhao, R.; Zhu, J.; Lu, X.; Li, Y.; Qiu, S.; Jia, L.; Jiao, X.; Song, S.; Fan, C.; Hao, R.; Song, H., Electrochemical DNA Biosensor Based on a Tetrahedral Nanostructure Probe for the Detection of Avian Influenza A (H7N9) Virus. ACS applied materials & interfaces 2015, 7, 8834-42. (25) Fan, J.; Liu, Y.; Xu, E.; Zhang, Y.; Wei, W.; Yin, L.; Pu, Y.; Liu, S., A label-free ultrasensitive assay of 8-hydroxy-2'-deoxyguanosine in human serum and urine samples via polyaniline deposition and tetrahedral DNA nanostructure. Anal. Chim. Acta 2016, 946, 48-55. (26) Ge, Z.; Lin, M.; Wang, P.; Pei, H.; Yan, J.; Shi, J.; Huang, Q.; He, D.; Fan, C.; Zuo, X., Hybridization chain reaction amplification of microRNA detection with a tetrahedral DNA nanostructure-based electrochemical biosensor. Anal. Chem. 2014, 86, 2124-30. (27) Li, Q.; Zhao, D.; Shao, X.; Lin, S.; Xie, X.; Liu, M.; Ma, W.; Shi, S.; Lin, Y., Aptamer-Modified Tetrahedral DNA Nanostructure for Tumor-Targeted Drug Delivery. ACS applied materials & interfaces 2017, 9, 36695-36701. (28) Li, Z.; Wei, B.; Nangreave, J.; Lin, C.; Liu, Y.; Mi, Y.; Yan, H., A replicable tetrahedral nanostructure self-assembled from a single DNA strand. J. Am. Chem. Soc. 2009, 131, 13093-8. (29) Liang, L.; Li, J.; Li, Q.; Huang, Q.; Shi, J.; Yan, H.; Fan, C., Single-particle tracking and modulation of cell entry pathways of a tetrahedral DNA nanostructure in live cells. Angew. Chem. Int. Ed. Engl. 2014, 53, 7745-50. (30) Miao, P.; Wang, B.; Chen, X.; Li, X.; Tang, Y., Tetrahedral DNA nanostructure-based microRNA biosensor coupled with catalytic recycling of the analyte. ACS applied materials & interfaces 2015, 7, 6238-43. (31) Peng, Q.; Shao, X. R.; Xie, J.; Shi, S. R.; Wei, X. Q.; Zhang, T.; Cai, X. X.; Lin, Y. F., Understanding the Biomedical Effects of the Self-Assembled Tetrahedral DNA Nanostructure on Living Cells. ACS applied materials & interfaces 2016, 8, 12733-9. (32) Shao, X.; Lin, S.; Peng, Q.; Shi, S.; Wei, X.; Zhang, T.; Lin, Y., Tetrahedral DNA Nanostructure: A Potential Promoter for Cartilage Tissue Regeneration via Regulating Chondrocyte Phenotype and Proliferation. Small 2017, 13. (33) Shi, S.; Peng, Q.; Shao, X.; Xie, J.; Lin, S.; Zhang, T.; Li, Q.; Li, X.; Lin, Y., Self-Assembled Tetrahedral DNA Nanostructures Promote Adipose-Derived Stem Cell Migration via lncRNA XLOC 010623 and RHOA/ROCK2 Signal Pathway. ACS applied materials & interfaces 2016, 8, 19353-63. (34) Zeng, D.; Wang, Z.; Meng, Z.; Wang, P.; San, L.; Wang, W.; Aldalbahi, A.; Li, L.; Shen, J.; Mi, X., DNA Tetrahedral Nanostructure-Based Electrochemical miRNA Biosensor for Simultaneous Detection of Multiple miRNAs in Pancreatic Carcinoma. ACS applied materials & interfaces 2017, 9, 24118-24125. (35) Li, C.; Faulkner-Jones, A.; Dun, A. R.; Jin, J.; Chen, P.; Xing, Y.; Yang, Z.; Li, Z.; Shu, W.; Liu, D.; Duncan, R. R., Rapid formation of a supramolecular polypeptide-DNA hydrogel for in situ three-dimensional multilayer bioprinting. Angew. Chem. Int. Ed. Engl. 2015, 54, 3957-61. (36) Xia, Z.; Wang, P.; Liu, X.; Liu, T.; Yan, Y.; Yan, J.; Zhong, J.; Sun, G.; He, D., Tumor-Penetrating Peptide-Modified DNA Tetrahedron for Targeting Drug Delivery. Biochemistry 2016, 55, 1326-31. (37) Zhang, Q.; Jiang, Q.; Li, N.; Dai, L. R.; Liu, Q.; Song, L. L.; Wang, J. Y.; Li, Y. Q.; Tian, J.; Ding, B. Q.; Du, Y., DNA Origami as an In Vivo Drug Delivery Vehicle for Cancer Therapy. ACS nano 2014, 8, 6633-6643.

18


Page 18 of 29

Page 19 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(38) Chen, F.; Bai, M.; Cao, K.; Zhao, Y.; Cao, X.; Wei, J.; Wu, N.; Li, J.; Wang, L.; Fan, C.; Zhao, Y., Programming Enzyme-Initiated Autonomous DNAzyme Nanodevices in Living Cells. ACS nano 2017, 11, 11908-11914. (39) Shi, S.; Lin, S.; Shao, X.; Li, Q.; Tao, Z.; Lin, Y., Modulation of chondrocyte motility by tetrahedral DNA nanostructures. Cell Prolif. 2017, 50. (40) Pei, H.; Zuo, X. L.; Zhu, D.; Huang, Q.; Fan, C. H., Functional DNA Nanostructures for Theranostic Applications. Acc. Chem. Res. 2014, 47, 550-559. (41) Liu, K.; Pan, D.; Wen, Y.; Zhang, H.; Chao, J.; Wang, L.; Song, S.; Fan, C.; Shi, Y., Identifying the Genotypes of Hepatitis B Virus (HBV) with DNA Origami Label. Small 2017. (42) Xie, X.; Liao, J.; Shao, X.; Li, Q.; Lin, Y., The Effect of shape on Cellular Uptake of Gold Nanoparticles in the forms of Stars, Rods, and Triangles. Sci. Rep. 2017, 7, 3827. (43) Liao, J.; Tian, T.; Shi, S.; Xie, X.; Ma, Q.; Li, G.; Lin, Y., The fabrication of biomimetic biphasic CAN-PAC hydrogel with a seamless interfacial layer applied in osteochondral defect repair. Bone research 2017, 5, 17018. (44) Brewer, G. J., Serum-Free B27/Neurobasal Medium Supports Differentiated Growth of Neurons from the Striatum, Substantia-Nigra, Septum, Cerebral-Cortex, Cerebellum, and Dentate Gyrus. J. Neurosci. Res. 1995, 42, 674-683. (45) Schulz, T. C.; Noggle, S. A.; Palmarini, G. M.; Weiler, D. A.; Lyons, I. G.; Pensa, K. A.; Meedeniya, A. C. B.; Davidson, B. P.; Lambert, N. A.; Condie, B. G., Differentiation of human embryonic stem cells to dopaminergic neurons in serum-free suspension culture. Stem Cells 2004, 22, 1218-1238. (46) Zhong, J.; Guo, B.; Xie, J.; Deng, S. W.; Fu, N.; Lin, S. Y.; Li, G.; Lin, Y. F.; Cai, X. X., Crosstalk between adipose-derived stem cells and chondrocytes: when growth factors matter. Bone research 2016, 4. (47) Zhang, Q.; Lin, S.; Zhang, T.; Tian, T.; Ma, Q.; Xie, X.; Xue, C.; Lin, Y.; Zhu, B.; Cai, X., Curved microstructures promote osteogenesis of mesenchymal stem cells via the RhoA/ROCK pathway. Cell Prolif. 2017, 50. (48) Ono, M.; Yin, P.; Navarro, A.; Moravek, M. B.; Coon, J. S. t.; Druschitz, S. A.; Serna, V. A.; Qiang, W.; Brooks, D. C.; Malpani, S. S.; Ma, J.; Ercan, C. M.; Mittal, N.; Monsivais, D.; Dyson, M. T.; Yemelyanov, A.; Maruyama, T.; Chakravarti, D.; Kim, J. J.; Kurita, T.; Gottardi, C. J.; Bulun, S. E., Paracrine activation of WNT/beta-catenin pathway in uterine leiomyoma stem cells promotes tumor growth. Proc. Natl. Acad. Sci. U. S. A. 2013, 110, 17053-8. (49) Tarapore, R. S.; Siddiqui, I. A.; Mukhtar, H., Modulation of Wnt/beta-catenin signaling pathway by bioactive food components. Carcinogenesis 2012, 33, 483-91. (50) Chung, H.; Li, E.; Kim, Y.; Kim, S.; Park, S., Multiple signaling pathways mediate ghrelin-induced proliferation of hippocampal neural stem cells. J. Endocrinol. 2013, 218, 49-59. (51) Chiang, M. Y.; Shestova, O.; Xu, L. W.; Aster, J. C.; Pear, W. S., Divergent effects of supraphysiologic Notch signals on leukemia stem cells and hematopoietic stem cells. Blood 2013, 121, 905-917. (52) Gao, F.; Zhang, Y. F.; Zhang, Z. P.; Fu, L. A.; Cao, X. L.; Zhang, Y. Z.; Guo, C. J.; Yan, X. C.; Yang, Q. C.; Hu, Y. Y.; Zhao, X. H.; Wang, Y. Z.; Wu, S. X.; Ju, G.; Zheng, M. H.; Han, H., miR-342-5p Regulates Neural Stem Cell Proliferation and Differentiation Downstream to Notch Signaling in Mice. Stem Cell Reports 2017, 8, 1032-1045. (53) Imayoshi, I.; Shimojo, H.; Sakamoto, M.; Ohtsuka, T.; Kageyama, R., Genetic visualization of notch signaling in mammalian neurogenesis. Cell. Mol. Life Sci. 2013, 70, 2045-57. (54) Katari, V.; Sen, D., NOTCH Signaling Is Essential for Maturation, Self-Renewal and Tri-Differentiation of In Vitro-Derived Human Neural Stem Cells. Mol. Ther. 2017, 25, 76-76. (55) Pierfelice, T.; Alberi, L.; Gaiano, N., Notch in the Vertebrate Nervous System: An Old Dog with New Tricks. Neuron 2011, 69, 840-855. (56) Jiang, D.; Sun, Y.; Li, J.; Li, Q.; Lv, M.; Zhu, B.; Tian, T.; Cheng, D.; Xia, J.; Zhang, L.; Wang, L.; Huang, Q.; Shi, J.; Fan, C., Multiple-Armed Tetrahedral DNA Nanostructures for Tumor-Targeting, Dual-Modality in Vivo Imaging.

19



ACS applied materials & interfaces 2016, 8, 4378-84. (57) Chao, J.; Liu, H. J.; Su, S.; Wang, L. H.; Huang, W.; Fan, C. H., Structural DNA Nanotechnology for Intelligent Drug Delivery. Small 2014, 10, 4626-4635. (58) Zhu, D.; Pei, H.; Yao, G. B.; Wang, L. H.; Su, S.; Chao, J.; Wang, L. H.; Aldalbahi, A.; Song, S. P.; Shi, J. Y.; Hu, J.; Fan, C. H.; Zuo, X. L., A Surface-Confined Proton-Driven DNA Pump Using a Dynamic 3D DNA Scaffold. Adv Mater 2016, 28, 6860-+. (59) Liu, B.; Song, C. Y.; Zhu, D.; Wang, X.; Zhao, M. Z.; Yang, Y. J.; Zhang, Y. N.; Su, S.; Shi, J. Y.; Chao, J.; Liu, H. J.; Zhao, Y.; Fan, C. H.; Wang, L. H., DNA-Origami-Based Assembly of Anisotropic Plasmonic Gold Nanostructures. Small 2017, 13.

20


Page 20 of 29

Page 21 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


TOC graphic

21



Figure 1. Successful synthesis and characterization of TDNs. A) Schematic diagram of TDNs. B) Confirmation of the successful synthesis of TDNs by 8% PAGE (TDN: red circle; Polymer: yellow circle); S1-2: S1+S2; S1-3: S1+S2+S3. C) Analysis images of TDNs by TEM (TDN: red triangle, Polymer: yellow circle). (n=3)

22


Page 22 of 29

Page 23 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 2. Undifferentiated cells authentication. Immunofluorescent images of NE-4C stem cells (Nestin: green, cytoskeleton: red, nucleus: blue). Scale bars are 25 µm.

23



Figure 3. NE-4C stem cells uptake of TDNs. A) Interaction of NE-4C stem cells with Cy5-ssDNA and Cy5-TDNs (Cy5: red, cytoskeleton: green, nucleus: blue). Scale bars are 25µm. B) Semi-quantitative analysis of fluorescence of Cy5-ssDNA and Cy5-TDNs. Data are presented as mean ± SD (n=4). Statistical analysis: *** p < 0.001.

24


Page 24 of 29

Page 25 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 4. Effects of TDNs on NE-4C stem cells proliferation and cell cycle. A) NE-4C stem cells proliferation analyzed by CCK-8 assay. Data are presented as mean ± SD (n = 4). Statistical analysis: * p < 0.05, ** p < 0.01. B) Percentage analysis of cell cycle distribution. Data are presented as mean ± SD (n = 4). Statistical analysis: ** p < 0.01, *** p < 0.001. C) Flow cytometry analysis of cell cycle after cells exposed to TDNs (250 nM) for 24 h. D) Analysis data of cell cycle distribution. Data are presented as mean ± SD (n = 4).

25



Figure 5. β-III-tubulin expression upon exposure to TDNs. A) Western blot analysis of β-III-tubulin protein expression level upon exposure to TDNs (250 nM) for 1, 3 and 7 days. B) Semi-quantitative analysis of western blot about β-III-tubulin protein expression level after cells treated with TDNs (250 nM) for 1, 3 and 7 days. Data are presented as mean ± SD (n = 4). Statistical analysis: **p < 0.01, *** p < 0.001. C) 26


Page 26 of 29

Page 27 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Quantitative real-time PCR analysis of β-III-tubulin gene expression upon exposure to TDNs (250 nM) for 1, 3 and 7 days. Data are presented as mean ± SD (n = 4). Statistical analysis: ** p < 0.01, *** p < 0.001. D) After treated with TDNs (250 nM) for 3 day, β-III-tubulin showed higher expression level in cells. Immunofluorescent images of cells treated with TDNs or without TDNs (β-III-tubulin: red, nucleus: blue). Scale bars are 25 µm. E) Quantification of average optical density of Figure 5D. Data are presented as mean ± SD (n = 4). Statistical analysis: ** p < 0.01. F) After treated with TDNs (250 nM) for 7 days, β-III-tubulin showed higher expression level in cells. Immunofluorescent images of cells treated with TDNs or without TDNs (β-III-tubulin: red, nucleus: blue). Scale bars are 25 µm. G) Quantification of average optical density of Figure 5E. Data are presented as mean ± SD (n = 4). Statistical analysis: ** p < 0.01.

27



Figure 6. A-C) Wnt pathway relevant proteins and genes expressed differently after cells exposed to TDNs (250 nM) for 24 h. D-F) Notch pathway relevant proteins and genes expressed differently after cells exposed to TDNs (250 nM) for 7 days. A) Western blot analysis of essential proteins expression level upon exposure to TDNs (250 nM) for 24 h. B) Semi-quantitative analysis of western blot about essential proteins expression level after cells treated with TDNs (250 nM) for 24 h. Data are presented as mean ± SD (n = 4). Statistical analysis: * p < 0.05, ** p < 0.01. C) Quantitative real-time PCR analysis of essential genes expression upon exposure to TDNs (250 nM) for 24 h. Data are presented as mean ± SD (n = 4). Statistical analysis: ** p < 0.01, *** p < 0.001. D) Western blot analysis of essential proteins expression level upon exposure to TDNs (250 nM) for 7 days. E) Semi-quantitative analysis of western blot about essential proteins expression level after cells treated 28


Page 28 of 29

Page 29 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


with TDNs (250 nM) for 7 days. Data are presented as mean ± SD (n = 4). Statistical analysis: **p < 0.01, *** p < 0.001. F) Quantitative real-time PCR analysis of essential genes expression upon exposure to TDNs (250 nM) for 7 days. Data are presented as mean ± SD (n = 4). Statistical analysis: ** p < 0.01, *** p < 0.001.

Figure 7. Schematic diagram showing the mechanism of TDNs enhanced the cells proliferation via Wnt/β-catenin pathway and neuronal differentiation via Notch pathway.

29


Self-Assembled Tetrahedral DNA Nanostructures Promote Neural

Recommend Documents