The Suppression Effects of Fat Mass and Obesity Associated Gene on the Hair Follicle-Derived Neural Crest Stem Cells Differentiating into Melanocyte by N6-Methyladenosine Modifying Microphthalmia-Associated Transcription Factor

Abstract

Background and objectives: Melanocyte (MC), derived from neural crest stem cell (NCSC), are involved in the production of melanin. The mechanism by which NCSC differentiates to MC remains unclear. N6-methyladenosine (m6A) modification was applied to discuss the potential mechanism.

Methods and results: NCSCs were isolated from hair follicles of rats, and were obtained for differentiation. Cell viability, tyrosinase secretion and activity, and transcription factors were combined to evaluated the MC differentiation. RT-qPCR was applied to determine mRNA levels, and western blot were used for protein expression detection. Total m6A level was measured using methylated RNA immunoprecipitation (MeRIP) assay, and RNA immunoprecipitation was used to access the protein binding relationship. In current work, NCSCs were successfully differentiated into MCs. Fat mass and obesity associated gene (FTO) was aberrant downregulated in MCs, and elevated FTO suppressed the differentiation progress of NCSCs into MCs. Furthermore, microphthalmia-associated transcription factor (Mitf), a key gene involved in MC synthesis, was enriched by FTO in a m6A modification manner and degraded by FTO. Meanwhile, the suppression functions of FTO in the differentiation of NCSCs into MCs were reversed by elevated Mitf.

Conclusions: In short, FTO suppressed the differentiating ability of hair follicle-derived NCSCs into MCs by m6A modifying Mitf.

Keywords: Differentiation; FTO; Melanocyte; Mitf; N6-methyladenosine; Neural crest stem cell.

Fig. 1

Neural crest origin and pluripotency of differentiation of NCSCs. (A) NCSCs in the suspended state. (B) NCSCs in the adherent state. (C) Gene expression profiles of NCSCs. Differentiated cells produce an immune response to (D) β-Tubulin III, (E) SMA and (F) tyrosinase.

Fig. 2

(A) Tyrosinase-stained cells were prominently increased and (B) the overall cell viability was markedly improved with the MC differentiation. **p<0.01.

Fig. 3

Identification of MCs differentiated from NCSCs. (A) Tyrosinase activity of MCs derived from NCSCs. (B) Protein levels of tyrosinase and Mitf determined by western blot. (C) mRNA levels of key genes in the formation MCs. (D) A series of recep-tors of melanogenic stimulants on MCs and transcription factors were accessed by RT-qPCR. *p<0.05, **p<0.01.

Fig. 4

mRNA of seven m6A-associated genes were accessed by RT-qPCR from primary NCSCs and differentiated MCs.

Fig. 5

FTO participates in the differentiation NCSCs into MCs. (A) mRNA level and (B) Protein expression of FTO in NCSCs with elevated FTO. (C) cell viability of MCs derived from NCSCs with elevated FTO and (D) Tyrosinase activity. (E) Protein levels of tyrosinase and Mitf determined by western blot. (F) mRNA levels of key genes in the formation MCs. (G) A series of receptors of melanogenic stimulants on MCs and transcription factors were accessed by RT-qPCR. *p<0.05, **p<0.01, ^#p<0.05, ^##p<0.01. FTO: fat mass and obesity-associated protein, NCSCs: neural crest stem cells, MCs: mela-nocytes.

Fig. 6

FTO can modify Mitf by m6A methylation to weaken the stability of Mitf. (A) Total m6A levels of melanogenic factors in MCs with elevated FTO. (B) Mitf enriched by FTO was accessed by RIP experiment method. (C, D) SRAMP was applied to predict the potential theoretical binding sites of Mitf for m6A modi-fication. (E) Luciferase activity of MCs tansfected with overexpression of FTO in both wild-type and mutant-type groups. (F) RT-qPCR assay was conducted to determine the expression of Mitf precursor and mature mRNA in MCs with elevated FTO. (G) The role of FTO in Mitf mRNA stability was assessed by using actinomycin D and RT-qPCR. **p<0.01.

Fig. 7

Mitf abolished the potency of FTO in MC formation. (A) mRNA level and (B) Protein expression of Mitf in NCSCs with elevated Mitf. (C) Tyrosinase activity and (D) Cell viability of MCs derived from NCSCs with elevated Mitf. (E) Protein levels of tyrosinase and Mitf determined by western blot. (F) mRNA levels of key genes in the formation MCs. (G) A series of receptors of melanogenic stimulants on MCs and transcription factors were accessed by RT-qPCR. *p<0.05, **p<0.01, compared with lv-nc and control groups. ^#p<0.05, ^##p<0.01, compared with FTO+lv-nc group.