MicroRNA regulation of neuron-like differentiation of adipose tissue-derived stem cells

Abstract

We have reported previously that adipose tissue-derived stem cells (ADSC) could be induced by isobutylmethylxanthine (IBMX) to differentiate into neuron-like cells and such differentiation was mediated by insulin-like growth factor I (IGF-I) signaling. In the present study we show that both IBMX and IGF-I upregulated neural markers beta-III-tubulin and paired-like homeobox transcription factor (Pitx3) in ADSC. Because Pitx3 is important for the maturation and function of dopaminergic neurons and has been shown to be regulated by microRNA miR-133b, we also examined the effects of IBMX and IGF-I on miR-133b expression. The results show that both IBMX and IGF-I upregulated miR-133b expression. When miR-133b was overexpressed in ADSC through transfection of a synthetic microRNA mimic, it caused a downregulation of beta-III-tubulin as evidenced by immunofluorescence staining and western blot analysis. Overexpression of miR-133b also downregulated Pitx3 and IGF-1 receptor (IGF-IR), and all such downregulation occurred at the protein but not the RNA level. The apparent posttranscriptional regulation was subsequently found to be exerted through a potential miR-133b target in the 3'-untranslated region of IGF-IR, as evidenced by luciferase assay. Thus, IGF-I signaling and miR-133b co-regulate potential neural differentiation of ADSC through a feedback mechanism, in which IGF-I upregulates miR-133b while miR-133b in turn downregulates IGF-IR.

Figure 1. IGF and IBMX upregulate β-III-tubulin and Pitx3 expression

ADSC were treated with the indicated reagents at top and then analyzed by western blotting for β-III-tubulin and Pitx3 expression, with β-actin serving as control. The experiment was repeated 3 times and each protein band analyzed by densitometry. Statistical analysis of the densitometric data is shown in panels B and C. “Relative expression” denotes ratio of β-III-tubulin or Pitx3 expression versus β-actin expression. * indicates significant difference (P <0.05) when compared to control. # indicates significant difference (P <0.05) when compared to corresponding treatment without PPP.

Figure 2. IGF and IBMX upregulate miR-133b expression

ADSC were treated with the indicated reagents at bottom and then analyzed by real-time PCR for miR-133b expression, which is presented as a ratio (relative expression) versus U6 RNA expression, based on 3 independent experiments. * indicates significant difference (P <0.05) compared to control. # indicates significant difference compared to corresponding treatment without PPP.

Figure 3. miR-133b overexpression downregulates β III-tubulin expression

(A) ADSC were uninduced (control) or induced with IBMX after being transfected with the indicated miRNAs. “Vehicle” denotes transfection reagent. The cells were then analyzed by immunofluorescence staining for β-III-tubulin expression (green). Cell nuclei were identified by DAPI staining (blue). Repeated 3 times. (B) ADSC were uninduced (control) or induced with IBMX after being transfected with the indicated miRNAs. “Vehicle” denotes transfection reagent. The cells were then analyzed by western blotting for β-III-tubulin expression, with β- actin serving as control. Repeated 3 times.

Figure 4. miR-133b overexpression downregulates β-III-tubulin, IGF-IR, and Pitx3 protein expression

(A) ADSC were uninduced (control) or induced with IBMX after being transfected with the indicated miRNAs. “Vehicle” denotes transfection reagent. The cells were then analyzed by western blotting for β-III-tubulin, IGF-IR and Pitx3 expression, with β-actin serving as control. Repeated 3 times. (B) ADSC were uninduced (control) or induced with IBMX after being transfected with the indicated miRNAs. “Vehicle” denotes transfection reagent. The cells were then analyzed by RT-PCR for β-III-tubulin and IGF-IR expression, with β-actin serving as control. Repeated 3 times. (C) ADSC were uninduced (control) or induced with IBMX after being transfected with the indicated miRNAs. “Vehicle” denotes transfection reagent. The cells were then analyzed by real-time PCR for Pitx3 expression, which is presented as a ratio (relative expression) versus NADPH expression, based on 3 independent experiments. * indicates significant difference (P <0.05) compared to control.

Figure 5. miR-133b overexpression downregulates IGF-IR posttranscriptionally

(A) COS7 cells were transfected with luciferase reporter vectors carrying wildtype (pMIR-IGF1R-3’UTR) of mutant (pMIR-IGF1R-3’UTR-M) version of the potential miR-133b target sequence in the 3’UTR of IGF-IR gene. The cells were also transfected with miR-133b (black bar) or miRcontrol (white bar) and pRL-TK (internal control expressing Renilla luciferase activity). The translational regulatory activity of the wildtype or mutant IGF-I 3’UTR was presented as a ratio (relative expression) between Firefly and Renilla luciferase activities, based on 3 independent experiments. * indicates significant difference (P <0.05) compared to miR-control. (B) ADSC were transfected with pMIR-IGF1R-3’UTR and pRL-TK, treated with the indicated reagents at bottom, and then analyzed for Firefly and Renilla luciferase activities. The translational regulatory activity of the 3’UTR of IGF-IR gene was presented as a ratio (relative expression) between Firefly and Renilla luciferase activities, based on 3 independent experiments. * indicates significant difference (P <0.05) compared to untreated control.