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. 2018 Dec;51(6):e12503.
doi: 10.1111/cpr.12503. Epub 2018 Aug 9.

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

Wenjuan Ma  1 , Xueping Xie  1 , Xiaoru Shao  1 , Yuxin Zhang  1 , Chenchen Mao  1 , Yuxi Zhan  1 , Dan Zhao  1 , Mengting Liu  1 , Qianshun Li  1 , Yunfeng Lin  1
Affiliations

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

Wenjuan Ma et al. Cell Prolif. 2018 Dec.

Erratum in

  • Corrigendum.
    [No authors listed] [No authors listed] Cell Prolif. 2021 Oct;54(10):e13120. doi: 10.1111/cpr.13120. Cell Prolif. 2021. PMID: 34605093 Free PMC article. No abstract available.

Abstract

Objectives: The main purpose of current study was to explore the effects of tetrahedral DNA nanostructures (TDNs) on neuroectodermal (NE-4C) stem cells migration and unveil the potential mechanisms.

Materials and methods: The successfully self-assembled TDNs were also determined by dynamic light scattering (DLS). A bidirectional wound-healing assay and transwell chamber assay were employed to test the migrating behaviour of NE-4C stem cells cultured under different conditions.

Results: Through an in vitro study, we found that stem cells could internalize TDNs quickly, and the cells' parallel and vertical migration was promoted effectively. Besides, the effects of TDNs were found being exerted by upregulating the gene and protein expression levels of RhoA, Rock2 and Vinculin, indicating that the RHOA/ROCK2 pathway was activated by the TDNs during the cell migration.

Conclusions: In conclusion, TDNs could enter NSCs without the aid of other transfection reagents in large amounts, whereas only small amounts of ssDNA could enter the cells. TDNs taken up by NSCs activated the RHOA/ROCK2 signalling pathway, which had effects on the relevant genes and proteins expression, eventually promoting the migration of NE-4C stem cells. These findings suggested that TDNs have great potential in application for the repair and regeneration of neural tissue.

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Conflict of interest statement

All authors declare no competing financial interest.

Figures

Figure 1
Figure 1
Successful synthesis and characterization of TDNs. A, Schematic diagram of TDNs. B, Confirmation of the successful synthesis of TDNs by 8% PAGE (polyacrylamide gel electrophoresis; TDNs: red circle). Lane 1 is S1. Lane 2 is S2. Lane 3 is S3. Lane 4 is S4. Lane 5 is S1+S2. Lane 6 is S1+S2+S3. Lane 7 is S1+S2+S3+S4, representing the successful synthesis of TDNs. C, Typical size distribution graph of TDN
Figure 2
Figure 2
Cellular uptake of TDNs. A, Interaction of NE‐4C stem cells with 250 nmol/L Cy5‐ssDNA (the control group) or 250 nM Cy5‐TDNs for 12 h (Cy5: red, cytoskeleton: green, nucleus: blue). Scale bars are 25 μm. B, Semi‐quantitative analysis of fluorescence of Cy5‐ssDNA and Cy5‐TDNs. Data are presented as mean ± SD (n = 4). Statistical analysis: ***P < .001. C, After treatment with 250 nmol/L Cy5‐ssDNA or 250 nmol/L Cy5‐TDNs for 12 h, flow cytometry examination and the analysis of cellular uptake of Cy5‐ssDNA and Cy5‐TDNs (negative control: treated with nothing; positive control: treated with Cy5‐ssDNA; TDNs: treated with Cy5‐TDNs). D, Semi‐quantitative analysis of the cellular uptake in flow cytometry. Data are presented as mean ± SD (n = 4). Statistical analysis: **P < .01, ***P < .001
Figure 3
Figure 3
A, Facilitated effects of TDNs on NE‐4C stem cells horizontal migration tested by a bidirectional wound‐healing assay at 0, 12 and 24 h. B, Histogram representation of the percentage of NE‐4C stem cells migration area at 0, 12 and 24 h on treatment without (the control group) or with TDNs. Data are presented as mean ± SD (n = 4). Statistical analysis: *P < .05, ***P < .001
Figure 4
Figure 4
A, Schematic diagram of the vertical cell migration tested by Transwell chamber assay. B, Fluorescence images showing the facilitated effects of TDNs on NE‐4C stem cell migration. The transmigrated NE‐4C stem cells’ nuclei were stained with DAPI (nucleus: blue). Scale bars are 100 μm. C, Cell number of transmigrated NE‐4C stem cells was presented by histogram. Data are presented as mean ± SD (n = 3). Statistical analysis: ***P < .001
Figure 5
Figure 5
A, Semi‐quantitative PCR of RhoA upon exposure to 250 nmol/L TDNs for 24 h. B, Semi‐quantitative analysis of semi‐quantitative PCR of about RhoA gene expression level. Data are presented as mean ± SD (n = 4). Statistical analysis: ***P < .001. C, Quantitative PCR of RhoA upon exposure to 250 nmol/L TDNs for 24 h. Data are presented as mean ± SD (n = 4). Statistical analysis: *P < .05. D, Western blotting analysis of RhoA protein expression level after treatment with 250 nmol/L TDNs for 24 h. E, Semi‐quantitative analysis of western blotting about RhoA protein expression level. Data are presented as mean ± SD (n = 4). Statistical analysis: ***P < .001. F, After treated with 250 nmol/L TDNs for 24 h, RhoA showed higher expression level in cells. Immunofluorescent images of cells treated with or without TDNs (RhoA: red, cytoskeleton: green, nucleus: blue). Scale bars are 25 μm. G, Semi‐quantitative analysis of average optical density of Figure 5F. Data are presented as mean ± SD (n = 4). Statistical analysis: ***P < .001
Figure 6
Figure 6
A, Semi‐quantitative PCR of Rock2 upon exposure to 250 nmol/L TDNs for 24 h. B, Semi‐quantitative analysis of semi‐quantitative PCR of about Rock2 gene expression level. Data are presented as mean ± SD (n = 4). Statistical analysis: **P < .01. C, Quantitative PCR of Rock2 upon exposure to 250 nmol/L TDNs for 24 h. Data are presented as mean ± SD (n = 4). Statistical analysis: ***P < .001. D, Western blotting analysis of Rock2 protein expression level after treatment with 250 nmol/L TDNs for 24 h. E, Semi‐quantitative analysis of western blotting about Rock2 protein expression level. Data are presented as mean ± SD (n = 4). Statistical analysis: ***P < .001. F, After treated with 250 nmol/L TDNs for 24 h, Rock2 showed higher expression level in cells. Immunofluorescent images of cells treated with or without TDNs (Rock2: red, cytoskeleton: green, nucleus: blue). Scale bars are 25 μm. G, Semi‐quantitative analysis of average optical density of Figure 6F. Data are presented as mean ± SD (n = 4). Statistical analysis: ***P < .001
Figure 7
Figure 7
A, Semi‐quantitative PCR of Vinculin upon exposure to 250 nmol/L TDNs for 24 h. B, Semi‐quantitative analysis of semi‐quantitative PCR of about Vinculin gene expression level. Data are presented as mean ± SD (n = 4). Statistical analysis: **P < .01. C, Quantitative PCR of Vinculin upon exposure to 250 nmol/L TDNs for 24 h. Data are presented as mean ± SD (n = 4). Statistical analysis: ***P < .001. D, Western blotting analysis of Vinculin protein expression level after treatment with 250 nmol/L TDNs for 24 h. E, Semi‐quantitative analysis of western blotting about Vinculin protein expression level. Data are presented as mean ± SD (n = 4). Statistical analysis: ***P < .001. F, After treated with 250 nmol/L TDNs for 24 h, Vinculin showed higher expression level in cells. Immunofluorescent images of cells treated with or without TDNs (Vinculin: red, cytoskeleton: green, nucleus: blue). Scale bars are 25 μm. G, Semi‐quantitative analysis of average optical density of Figure 7F. Data are presented as mean ± SD (n = 4). Statistical analysis: ***P < .001
Figure 8
Figure 8
Schematic diagram showing the underlying mechanism of TDNs promoted the NE‐4C stem cells migration via activating RHOA/ROCK2 pathway
See this image and copyright information in PMC

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References

    1. Hoffman RM. Nestin‐expressing hair follicle‐accessible pluripotent stem cells for nerve and spinal cord repair. Cells Tissues Organs. 2014;200:42‐47. - PubMed
    1. Kim SU, Lee HJ, Kim YB. Neural stem cell‐based treatment for neurodegenerative diseases. Neuropathology. 2013;33:491‐504. - PubMed
    1. Nabetani M, Shintaku H, Hamazaki T. Future perspectives of cell therapy for neonatal hypoxic‐ischemic encephalopathy. Pediatr Res. 2017;83:356‐363. - PubMed
    1. Niimi Y, Levison SW. Pediatric brain repair from endogenous neural stem cells of the subventricular zone. Pediatr Res. 2017;83:385‐396. - PubMed
    1. Yue Y, Yang X, Zhang L, et al. 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. - PMC - PubMed

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