MiR-26a-tetrahedral framework nucleic acids mediated osteogenesis of adipose-derived mesenchymal stem cells

Abstract

Objectives: Delivery systems that provide time and space control have a good application prospect in tissue regeneration applications, as they can effectively improve the process of wound healing and tissue repair. In our experiments, we constructed a novel micro-RNA delivery system by linking framework nucleic acid nanomaterials to micro-RNAs to promote osteogenic differentiation of mesenchymal stem cells.

Materials and methods: To verify the successful preparation of tFNAs-miR-26a, the size of tFNAs-miR-26a were observed by non-denaturing polyacrylamide gel electrophoresis and dynamic light scattering techniques. The expression of osteogenic differentiation-related genes and proteins was investigated by confocal microscope, PCR and western blot to detect the impact of tFNAs-miR-26a on ADSCs. And finally, Wnt/β-catenin signaling pathway related proteins and genes were detected by confocal microscope, PCR and western blot to study the relevant mechanism.

Results: By adding this novel complex, the osteogenic differentiation ability of mesenchymal stem cells was significantly improved, and the expression of alkaline phosphatase (ALP) on the surface of the cell membrane and the formation of calcium nodules in mesenchymal stem cells were significantly increased on days 7 and 14 of induction of osteogenic differentiation, respectively. Gene and protein expression levels of ALP (an early marker associated with osteogenic differentiation), RUNX2 (a metaphase marker), and OPN (a late marker) were significantly increased. We also studied the relevant mechanism of action and found that the novel nucleic acid complex promoted osteogenic differentiation of mesenchymal stem cells by activating the canonical Wnt signaling pathway.

Conclusions: This study may provide a new research direction for the application of novel nucleic acid nanomaterials in bone tissue regeneration.

FIGURE 1

Successful synthesis of tFNAs–miR‐26a. (A) Sketch map of tFNAs–miR‐26a. (B) Confirmation of the successful synthesis of tFNAs–miR‐26a by native SDS‐PAGE (M: marker, S: single‐stranded DNA). (C) Confirmation of the successfully assembled tFNAs–miR‐26a by dynamic light scattering.

FIGURE 2

Cellular uptake of tFNAs–miR‐26a. (A) Cellular uptake of Cy5‐tFNAs–miR‐26a, Cy5‐tFNAs, and Cy5‐single‐stranded DNA in ADSCs (Cytoplasm: green; Nucleus: blue; Cy5: red). Scale bars are 50 μm. (B) Cellular uptake of Cy5‐tFNAs–miR‐26a, Cy5‐tFNAs, and Cy5‐single‐stranded DNA by flow cytometry.

FIGURE 3

Enhancement of ALP activity and calcium nodules formation after exposure to tFNAs–miR‐26a. (A) Osteogenic differentiation was detected by ALP staining (top) at day 7 and NBT‐formation in ALP‐stained cells after 7 days of osteogenic differentiation (bottom). (B) Osteogenic differentiation was detected by Alizarin Red staining (top) at day 15 and calcium nodules in Alizarin Red‐stained cells after osteogenic induction for 15 days (bottom).

FIGURE 4

Detection of osteogenic differentiation‐specific proteins and genes. (A) Western blot analysis of protein expression levels upon exposure to tFNAs–miR‐26a (250 nM) for 24 h. (B) Quantification of protein expression levels upon exposure to tFNAs–miR‐26a (250 nM) for 24 h. Data are presented as mean ± SD (n = 4). Student's t test: **p < 0.01, ***p < 0.001. (C) Quantification of gene expression levels upon exposure to tFNAs–miR‐26a (250 nM) for 24 h. Data are presented as mean ± SD (n = 4). Student's t test: **p < 0.01, ***p < 0.001. (D) Photomicrographs showing treated ADSCs (Cytoplasm: green, Nucleus: blue, Runx2: red). Scale bars are 50 μm. (E) Photomicrographs showing treated ADSCs (Cytoplasm: green, Nucleus: blue, OPN: red). Scale bars are 50 μm.

FIGURE 5

Detection of canonical Wnt/β‐catenin signaling pathway related proteins and genes. (A) Western blot analysis of protein expression levels upon exposure to tFNAs–miR‐26a (250 nM) for 24 h. (B) Quantification of protein expression levels upon exposure to tFNAs–miR‐26a (250 nM) for 24 h. Data are presented as mean ± SD (n = 4). Student's t test: **p < 0.01, ***p < 0.001. (C) Quantification of gene expression levels upon exposure to tFNAs–miR‐26a (250 nM) for 24 h. Data are presented as mean ± SD (n = 4). Student's t test: **p < 0.01, ***p < 0.001. (D) Photomicrographs showing treated ADSCs (cytoplasm: green, nucleus: blue, GSK‐3β: red). Scale bars are 50 μm. (E) Photomicrographs showing treated ADSCs (Cytoplasm: green, Nucleus: blue, β‐Catenin: red). Scale bars are 50 μm.