Tetrahedral DNA nanostructures facilitate neural stem cell migration via activating RHOA/ROCK2 signalling pathway

2.1. Cell culture

NE‐4C stem cells of mouse were bought from ATCC (CRL‐2925, VA, USA). Dulbecco's modified Eagle's medium (DMEM, Gibco, Grand Island, USA) was used to culture the cells which containing 1% (v/v) Penicillin‐streptomycin solution (Gibco, Grand Island, USA), and 10% (v/v) foetal bovine serum (FBS, Corning, New York, USA). The cells were cultivated in a humidified incubator at the temperature of 37°C, 5% CO₂ in the atmosphere. The growth medium was changed thrice every week.

2.2. TDNs synthesis and characterization

The same concentrations of each ssDNAs (TaKaRa, Ostu, Japan) were added to TM buffer (Tris‐HCl = 10 mmol/L; MgCl₂ = 50 mmol/L, pH = 8.0; Bio‐Rad, Hercules, USA). The mixtures were heated for 10 minutes at 95°C and then cooled at 4°C for 40 minutes. TDNs’ concentration used in our experiment is 250 nmol/L, which was proven to be the optimal concentration in our previous studies.40, 41, 43

To ensure the synthesis of self‐assembled TDNs, we tested the size of the TDNs and the 4 ssDNA by Polyacrylamide gel electrophoresis (8% PAGE, Beyotime, Nanjing, China). The successfully self‐assembled TDNs were also determined by dynamic light scattering (DLS) in ddH₂O at 250 nmol/L using a ZETAPals analyser (Brookhaven Instruments, Holtsville, NY, USA).

2.3. Cellular uptake of TDNs

To ensure that the cells could take in TDNs, we treated the cells with TDNs or ssDNA, which were both modified with Cyanine‐5 (Cy5). First, cells were seeded onto confocal dishes. The growth medium was changed to a medium with Cy5‐TDNs or Cy5‐ssDNA for 12 hours. Then, we washed the cell samples using phosphate‐buffered saline (PBS, Gibco, Grand Island, USA) for 3 times, and fixed them with paraformaldehyde solution (4% w/v, Boster, Wuhan, China). After all samples were rinsed 3 times, FITC‐labelled phalloidin (Sigma, St Louis, USA), together with DAPI (Sigma, St Louis, USA), was applied to stain each of cytoskeleton and nucleus. All samples were washed by PBS 3 times before being imaged under a confocal laser microscope (Nikon N‐SIM, Japan).

Cellular uptake of TDNs was also tested by flow cytometry. Briefly, cells were firstly plated to 6‐well plate (2 × 10⁵/well); then, we changed the medium with that containing Cy5‐TDNs or Cy5‐ssDNA. After 12 hours, the cells were collected and tested by the flow cytometer (FC500 Beckman, Brea, USA).

2.4. Parallel cell migration (wound healing assay)

A bidirectional wound‐healing assay was employed to test the migrating behaviour of NE‐4C stem cells cultured under different conditions. Briefly, cells were first plated to 6‐well plates and then incubated at 5% CO₂ and 37°C. Sterilized pipette tips were applied to form a bidirectional wound by scratching the single‐layer cells. PBS was applied to wash the cell debris away. Cells were treated with medium without FBS (the control groups: without TDNs) and the same medium containing TDNs (the experimental groups: 250 nmol/L TDNs). Wound closure of cells was imaged after incubation for 0, 12 and 24 hours.

2.5. Vertical cell migration (Transwell chamber assay)

Transwell insert (pore diameter: 8 μm; Corning) was applied to analyse the vertical cell migration.54 Transwell inserts were attached to 6‐well culture plate, and cells (1 × 10⁵/well) were seeded on the upper half of the insets. The growth medium was changed to the medium without FBS (the control groups: without TDNs; the experimental groups: 250 nmol/L TDNs). After incubated for 24 hours, cells in the upper insets were fixed in paraformaldehyde, staining with DAPI. The amount of cells presented in the lower part was determined by manual counting, and the results of migration were presented by using the mean number of cells.

2.6. Semi‐quantitative PCR and quantitative PCR

Total RNA of cells treating with or without TDNs was extracted by an RNeasy Plus Mini Kit and treated with a genomic DNA eliminator. The RNA samples were reverse transcribed into cDNA using a synthesis kit (TaKaRa, Ostu, Japan). The mRNAs (Table 1) expressions of each group were normalized to the housekeeper genes of glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) and evaluated. Semi‐quantitative PCR was conducted with a PCR kit (Mbi, TaKaRa, Ostu, Japan), while quantitative PCR (qPCR) was conducted using SYBR Green II PCR master mix (TaKaRa, Ostu, Japan) and a Bio‐Rad CFX96TM thermal cycler (Bio‐Rad). The semi‐quantitative PCR procedure included a 30‐second denaturation at 94°C, 30‐second annealing cycle at 55‐65°C and a 30‐second elongation cycle at 72°C, for 28‐35 amplification cycles. Products were detected by 2% agarose gel electrophoresis in TAE buffer and stained using Gel‐Red (Sigma, St Louis, USA). The procedure for qPCR comprised 2 minutes of denaturation at 95°C, 5 seconds annealing at 95°C and 30 seconds elongation at 60°C for 40 cycles.

2.7. Western blotting

After 24 hours treated with 250 nmol/L TDNs or vehicle control, NE‐4C stem cells were washed with PBS after thrice loop operations, and a Whole Cell Lysis Assay (KeyGEN Institute of Biotechnology, Jiangsu, China) was applied to harvest all the proteins. The protein samples and 5× loading buffer (Beyotime, Nanjing, China) were mixed together at a ratio of 4:1, and then the mixed samples were heated for 3 minutes at 100°C. The mixed samples were subjected to SDS‐PAGE (Beyotime, Nanjing, China) at different gel concentrations to separate the proteins, and then transferred to Polyvinylidene fluoride membranes (PVDF, TaKaRa, Ostu, Japan). After incubating with 5% skim milk powder solution for 1 hour, all membranes were incubated with primary antibodies (anti‐GAPDH, anti‐RhoA, anti‐Rock2, and anti‐Vinculin; Abcam, Cambridge, U.K) overnight at 4°C. All membranes were rinsed with TBST thrice next day and incubated with secondary antibodies for 1 hour. All membranes were washed thrice with TBST and tested by an enhanced chemiluminescence detection system (Bio‐Rad).

2.8. Immunofluorescence staining

Cells in confocal dishes treated with or without TNDs for 24 hours were fixed in paraformaldehyde solution. All samples were then treated with 0.5% Triton X‐100 and then blocked with 5% sheep serum. The samples were treated overnight with primary antibodies solution (anti‐RhoA, anti‐Rock2 and anti‐Vinculin; Abcam, Cambridge, U.K) at 4°C. The next day, the cells were incubated with secondary antibodies for 1 hour. The cytoskeleton was stained with phalloidine at 37°C for 1 hour and the nuclei with DAPI for 10 minutes. Every step in this experiment was followed by 3 washes with PBS for 10 minutes. Finally, a confocal laser microscope (Nikon N‐SIM, Japan) was applied to capture images.

2.9. Statistical analysis

SPSS (version 23.0) was applied for data analysis. Intergroup differences were compared using analysis of variance (ANOVA) and t test, and P < .05 indicated that the test results were statistically significant. All experiments and analyses were conducted in triplicate and replicated more than 3 times.

3.1. TDN characterizations

TDNs comprised 4 different ssDNA (Figure 1A), and each ssDNA was designed with a specific sequence based on the base complementation pairing rules (Table 2). To evaluate the synthesis and characterization of the TDNs, 8% PAGE was performed to assess the size and size distribution of the TDNs. Figure 1B shows that the TDNs were successfully synthesized. Figure 1C shows that the average size of TDNs was 16.801 ± 0.237 nm. These results indicated that the TDNs were successfully constructed.

3.2. Cellular uptake of TDNs

To ensure that NE‐4C stem cells could take in abundant TDNs, but not ssDNA, fluorescence staining and flow cytometry were used to track TDNs and ssDNA. As shown in Figure 2A,B, the fluorescence signal of Cy5 in the cells treated with Cy5‐loaded TDNs was stronger than that from cells treated with Cy5‐loaded ssDNA. Flow cytometry showed that more cells took in Cy5‐loaded TDNs than Cy5‐loaded ssDNA (Figure 2C‐D). These results indicated that the cells could take in TDNs specifically, which is a prerequisite of TDNs to affect cellular biological behaviour.

3.3. Changes in NE‐4C stem cell migration

To observe TDNs’ actions of horizontal migration on cells, we used 250 nmol/L TDNs to treat cells and analysed the migration by a wound‐healing assay, which has been applied to detect the cell migration in vitro widely. Cells migrated towards the wound areas for 24 hours, and the control group cells migrated markedly slower than the groups cells treated with 250 nmol/L TDNs (Figure 3A). Statistical analysis of the area of wound healing showed that NE‐4C stem cells migration was promoted significantly after treatment with TDNs (Figure 3B).

To further confirm that TDNs could affect cell vertical migration, Transwell chamber assay was used. According to Figure 4A,B, the stem cells were plated onto the upper chamber of Transwell and treated with or without 250 nmol/L TDNs for 24 hours. Compared with the control groups, more cells of the experimental groups incubated with 250 nmol/L TDNs, migrated onto the lower surface of the Transwell. Moreover, the quantitative analysis shown in Figure 4C indicated that TDNs could enhance the migration of NE‐4C stem cells significantly. These results showed that TDNs could facilitate the stem cell migration, which is a prerequisite for the regeneration of the injured nervous system.

3.4. TDNs promote cell migration via the RHOA/ROCK2 pathway

Previous studies demonstrated that the RHOA/ROCK2 pathway is closely related to cell migration; therefore, changes in the expressing levels of ras homologue family member A (RhoA), rho associated coiled‐coil containing protein kinase 2 (Rock2) and Vinculin were tested in this study. From the results of semi‐quantitative PCR (Figures 5A‐B, 6A‐B, and 7A‐B) and quantitative PCR (Figures 5C, 6C and 7C), the expressions of RhoA, Rock2 and Vinculin in cells with the treatment of TDNs for 24 hours were activated by comparison with the control group.

To further explore the mechanism by which TDNs activate the RHOA/ROCK2 pathway, we used both western blotting and immunofluorescence staining to analyse the protein levels of RhoA, Rock2 and Vinculin. From the results of western blotting, the group with TDNs had higher levels of RhoA, Rock2 and Vinculin than the control group (Figures 5D‐E, 6D‐E and 7D‐E); meanwhile, the immunofluorescence staining intensities of the 3 proteins in cells treated with TDNs were much stronger than those in the cells of the control group (Figures 5F,G, 6F,G and 7F,G). The gene expression and protein analysis results were consistent, thus indicated that TDNs could accelerate cells migration by upregulating RHOA/ROCK2 signalling pathway (Figure 8).