MicroRNAs regulate synthesis of the neurotransmitter substance P in human mesenchymal stem cell-derived neuronal cells

Abstract

MicroRNAs (miRNAs) are a class of 19- to 23-nt, small, noncoding RNAs, which bind the 3' UTR of target mRNAs to mediate translational repression in animals. miRNAs have been shown to regulate developmental processes, such as self-renewal of stem cells, neuronal differentiation, myogenesis, and cancer. A functional role of miRNAs in the regulation of neurotransmitter synthesis has yet to be ascribed. We used mesenchymal stem cells (MSCs) as a model to study miRNA-mediated neurotransmitter regulation in developing neuronal cells. MSCs are mesoderm-derived cells, primarily resident in adult bone marrow, which can generate functional neuronal cells. We have previously shown that human MSC-derived neuronal cells express the neurotransmitter gene, Tac1, but do not synthesize the gene's encoded peptide, the neurotransmitter substance P (SP), unless stimulated with the inflammatory mediator IL-1alpha. These findings suggested a potential role for miRNAs in the regulation of SP synthesis. Here, we report on the miRNA profile of undifferentiated human MSCs and MSC-derived neuronal cells by using miRNA-specific bioarrays. miRNAs that were increased in the neuronal cells and decreased after IL-1alpha stimulation were analyzed by the miRanda algorithm to predict Tac1 mRNA targets. Putative miR-130a, miR-206, and miR-302a binding sites were predicted within the 3' UTR of Tac1. Target validation using a luciferase reporter system confirmed the miR-130a and miR-206 sites. Specific inhibition of miR-130a and miR-206 in the neuronal cells resulted in SP synthesis and release. The studies provide a different approach in ascribing a new regulatory role for miRNAs in regulating neurotransmitter synthesis.

Fig. 1.

Cartoon depicting the mechanism of miRNA transcription, processing, and regulatory activity. miRNA genes are transcribed by RNA polymerase II to form primary miRNA (pri-miRNA) molecules. The ribonuclease, Drosha, then cleaves the pri-miRNA to release the pre-miRNA for cytoplasmic export and processing by Dicer. The mature miRNA product associates with the RNA-induced silencing complex for loading onto the 3′ UTR of target mRNAs to mediate translational repression.

Fig. 2.

miRNA and SP levels in D0 and D12 cells with or without IL-1α stimulation. (A and B) Fold change in candidate miRNAs (miR-130a, miR-206, and miR-302a) (A) and U6 small nuclear RNA in D0 and D12 cells (B) with or without IL-1α stimulation. (C and D) Candidate miRNAs were re-examined at D6 and D10 (C) and in D12 cells stimulated with various concentrations of IL-1α (D). All RNA levels were determined by real-time RT-PCR. Results are presented as mean fold change ± SD; n = 5. Normalizations with 5S rRNA were arbitrarily assigned values of 1. (E) D12 cells were stimulated for 16 h with various concentrations of IL-1α, and SP release was quantified by ELISA. Results are presented as mean ± SD; n = 5. *, P < 0.05 vs. D0; **, P < 0.05 vs. unstimulated D12 cells; ***, P < 0.05 vs. D12 cells stimulated with 0.01 ng/ml IL-1α.

Fig. 3.

Target validation of candidate miRNAs and effects of IL-1α on transfected pre-miRs. (A) The Tac1 3′ UTR was cloned into the pMIR-REPORT miRNA luciferase reporter system (pMIR-R/Tac1/SG). (B) D0 cells were cotransfected with pMIR-R/Tac1/SG and candidate pre-miRs (miR-130a, miR-206, and/or miR-302a), and luciferase and β-gal activities were measured. In parallel studies, uninduced cells were transfected with pre-miR negative control. Results are presented as the mean ± SD of normalized luciferase; n = 5. Normalizations were performed with luciferase/β-gal activities in cells transfected with pMIR-R/Tac1/SG alone, arbitrarily assigning a value of 1,500. (C) D0 cells were transfected with candidate or negative control pre-miRs or left untransfected. Cells were then stimulated for 16 h with IL-1α or unstimulated, and candidate miRNA levels were determined by real-time RT-PCR. Results are presented as mean fold change ± SD; n = 5. Normalizations with 5S rRNA were arbitrarily assigned values of 1. (D) D0 cells were prepared as in C, and SP levels were quantified by ELISA. Results are presented as mean ± SD; n = 5. *, P < 0.05 vs. negative control cells; **, P < 0.05 vs. untransfected cells stimulated with IL-1α; ***, P < 0.05 vs. untransfected, unstimulated cells.

Fig. 4.

Synergistic translational repression of Tac1 mRNA by miR-130a and miR-206. (A) D0 and D12 cells were transfected with anti-miR-130a, anti-miR-206, anti-miR-302a, or negative control. Endogenous miRNA levels were determined by real-time RT-PCR. Results are presented as mean ± SD percent expression; n = 5. Normalizations were performed with 5S rRNA in negative control cells, arbitrarily assigning a value of 100%. (B) D12 cells were transfected with anti-miR-130a, anti-miR-206, and/or anti-miR-302a or negative control, and SP release was quantified by ELISA. D12 cells stimulated for 16 h with IL-1α served as positive control. Results are presented as mean ± SD; n = 5. (C) D0 and D12 cells were transfected with anti-miR-130a, anti-miR-206, and anti-miR-302a or negative control, and Tac1 mRNA expression was determined by real-time RT-PCR. Results are presented as mean ± SD fold change; n = 5. Normalizations were performed with 5S rRNA in negative control cells, arbitrarily assigning a value of 1. *, P < 0.05 vs. negative control cells; **, P < 0.05 vs. D0 cells.