Increased levels of miR-124 in human dental pulp stem cells alter the expression of neural markers

Abstract

Auditory neuropathy is the particular form of deafness in humans which cannot be treated by replacement therapy. Human dental pulp stem cells (hDPSCs) are derived from an ectomesenchymal neural crest cell population. Therefore, they possess a promising capacity for neuronal differentiation and repair. miR-124, a key regulator of neuronal development in the inner ear, is expressed at high levels in auditory and vestibular neurons. Here, we evaluated the possible effect of miR-124 in alteration of neural protein markers expression. Using quantitative reverse transcription-PCR (qRT-PCR) analyses and immunofluorescence staining, we studied the expression patterns of neural progenitor markers (Nestin, NOTCH1, and SOX2) and neural markers (β-tubulin III, GATA-3, and peripherin) upon transfection of hDPSCs with miR-124. The qRT-PCR results showed that Nestin was upregulated 6 h post-transfection. In contrast, Nestin expression exhibited a decreasing trend 24 h and 48 h post-transfection. Higher levels of β-tubulin III, 6 h and 16 h post transfection in RNA level as compared with control cells, were determined in transfected DPSCs. However, β-tubulin-III expression decreased 48 h post-transfection. The immunoflourescence results indicated that transfection of hDPSCs with miR-124, only affected Nestin among the studied neural progenitor and neural marker expression in protein level.

Keywords: DPSCs; Nestin; Sensorineural hearing loss; Spiral ganglion neurons; basic fibroblast growth factor, bFGF; bone morphogenetic protein 4, BMP4; bovin serum albumin, BSA; brain derived neurotrophic factor, BDNF; epidermal growth factor, EGF; human dental pulp stem cells, hDPSCs; miR-124; neurotrophin-3, NT3; quantitative reverse transcription-PCR, qRT-PCR; sonic hedgehog, SHH; spiral ganglion neurons, SGNs.

Fig. 1

qRT-PCR analysis of the miR-124 level in DPSCs after transfection (6 h, 16 h, 24 h, and 48 h). As shown by the data, the highest increase in the miR-124 level occurred 6 h post-transfection as compared with control cells (Mann–Whitney, P = 0.004). The data were normalized to expression levels in the control.

Fig. 2

qRT-PCR analysis of Nestin mRNA expression in DPSCs post-transfection. As shown by the data, Nestin mRNA expression was upregulated 6 h post-transfection as compared with that of the control (Mann–Whitney, P = 0.004) and downregulated 24 h (Mann–Whitney, P = 0.029) and 48 h (Mann–Whitney, P = 0.004) post-transfection. No statistically significant alteration in Nestin mRNA expression was observed 16 h post-transfection. The data were normalized to expression levels in the control.

Fig. 3

Immunofluorescence analysis of Nestin (A), NOTCH1 (B), and SOX2 (C) expression in DPSCs 6 h after transfection with miR-124 (A1,2,3; B1,2,3; C1,2,3) versus scrambled miR (A4,5,6; B4,5,6; C4,5,6). As shown by these figures, an increased level of miR-124 6 h post-transfection affected the expression of Nestin but not that of NOTCH1 or SOX2.

Fig. 4

qRT-PCR analyses of β-tubulin III, GATA-3, and peripherin mRNA expression in DPSCs post-transfection. The data show increased β-tubulin III mRNA expression 6 h (Mann–Whitney, P = 0.005) and 16 h (Mann–Whitney, P = 0.008) post-transfection as compared with that in the control (A). There was no statistically significant alteration in GATA-3 (B) and peripherin (C) mRNA expression in the scrambled control versus miR-124 transfected cells in any post-transfection hours.

Fig. 5

Immunofluorescence analyses of β-tubulin III (A), GATA-3 (B), and peripherin (C) expression in DPSCs 6 h after transfection with miR-124 (A1,2,3; B1,2,3; C1,2,3) versus scrambled miR (A4,5,6; B4,5,6; C4,5,6). As shown by the figures, the immunofluorescence analysis revealed no significant alteration in the expression of β-tubulin III, GATA3 and Peripherin upon hDPSC transfection with miR-124 6 h post-transfection (however the results of qRT-PCR revealed upregulation of the neuron-specific marker β-tubulin III at the mRNA level).