Int J Stem Cells.  2021 Nov;14(4):434-446. 10.15283/ijsc20118.

MiR-148a-3p Regulates the Invasion and Odontoblastic Differentiation of Human Dental Pulp Stem Cells via the Wnt1/β-Catenin Pathway

• Li Q
• Huang L

• KMID: 2522562
• DOI: http://doi.org/10.15283/ijsc20118

Abstract

Background and Objectives
MiR-148a-3p has been reported to regulate the differentiation of marrow stromal cell osteoblast. In this study, whether miR-148a-3p regulated the odontoblastic differentiation of human dental pulp stem cells (hDPSCs) or not was explored.
Methods and Results
The hDPSCs were isolated and identified via flow cytometry. Targets of miR-148a-3p were identi-fied via bioinformatics and dual-luciferase reporter assay. After the cell was cultured in the odontogenic differentiation medium or infected, cell viability, invasion, and odontoblastic differentiation were detected via MTT, transwell, and Alizarin Red S staining, respectively. The miR-148a-3p, Wnt1, β-catenin, DSPP, DMP-1, RUNX2, OCN, and Smad4 expressions were determined by RT-qPCR and Western blot. The hDPSCs odontoblastic differentiation downregulated the miR-148a-3p expression and upregulated Wnt1 expression. Wnt1 was determined as the target for miR-148a-3p. MiR-148a-3p mimic and siWnt1 suppressed the cell viability, invasion, and odontoblastic differentiation of hDPSCs and inhibited the Wnt1, β-catenin, DSPP, DMP-1, RUNX2, OCN, and Smad4 expressions. In contrast, miR-148a-3p inhibitor and overexpressed Wnt1 promoted the cell viability, invasion, and odontoblastic differentiation of hDPSCs, and upregulated the Wnt1, β-catenin, DSPP, DMP-1, RUNX2, OCN, and Smad4 expressions. Also, miR-148a-3p mimic and inhibitor reversed the effects of Wnt1 overexpression and siWnt1.
Conclusions
MiR-148a-3p modulated the invasion and odontoblastic differentiation of hDPSCs through the Wnt1/β-catenin pathway.

Keyword

Human dental pulp stem cells; miR-148a-3p; Odontoblastic differentiation; Wnt1; β-catenin

Figure

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  Fig. 1 hDPSCs was isolated and identified. (A) Cell morphology of hDPSCs was observed. (B) The levels of the biomarkers in hDPSCs were detected by flow cytometry. All experiments were conducted in triplicate (hDPSCs: human dental pulp stem cells).

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  Fig. 2 The expression of miR-148a-3p was decreased and the expre-ssion of Wnt1 was increased after the differentiation of hDPSCs. (A) The formation of calcium nodules in hDPSCs after being cultured for 3, 7 and 14 d was detected by Alizarin red staining. (B) The expression of miR-148a-3p was detected by RT-qPCR. U6 used as an internal control. (C) The expression of Wnt1 was detected by RT-qPCR. GAPDH used as an internal control. All experiments were conducted in triplicate (**p<0.01, ***p<0.001, vs. 3 d) (hDPSCs: human dental pulp stem cells).

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  Fig. 3 MiR-148a-3p mimic inhibited the cell viability, invasion, and differentiation of hDPSCs by down-regulating the expressions of DSPP, DMP-1, RUNX2, OCN, and Smad4. (A) The transfection efficiency of miR-148a-3p mimic was detected by RT-qPCR. U6 was used as an internal control. (B) The cell viability of hDPSCs was detected by MTT assays. (C, E) The invasion of hDPSCs was detected by Transwell assays. (D) The formation of calcium nodules in hDPSCs after being cultured for 14 days was detected by Alizarin red staining. (F) The expressions of DSPP, DMP-1, RUNX2, OCN, and Smad4 in hDPSCs were detected by RT-qPCR. GAPDH was used as an internal control. (G, H) The protein expressions of DSPP, DMP-1, RUNX2, OCN, and Smad4 in hDPSCs were detected by Western blot. GAPDH was used as an internal control. All experiments were conducted in triplicate (*p< 0.05, **p<0.01, ***p<0.001, vs. MC) (hDPSCs: human dental pulp stem cells, MC: mimic control, M: miR-148a-3p mimic).

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  Fig. 4 MiR-148a-3p specifically targets Wnt1. (A) Wnt1-3’-UTR contains a binding site of miR-148a-3p. (B) Dual-luciferase reporter assay validated that miR-148a-3p targeted Wnt1 in hDPSCs. Luciferase from firefly was used as reporter gene and luciferase from sea kidney as internal reference gene. All experiments were conducted in triplicate (***p<0.001, vs. MC) (hDPSCs: human dental pulp stem cells, MC: mimic control, M: miR-148a-3p mimic).

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  Fig. 5 MiR-148a-3p reversed the regulatory effect of Wnt1 on the cell viability, invasion, and differentiation of hDPSCs. (A) Infection efficiency of Wnt1 and siWnt1 was detected by RT-qPCR. GAPDH was used as an internal control. (B) Transfection efficiency of miR-148a-3p inhibitor was detected by RT-qPCR. U6 was used as an internal control. (C, D) The cell viability of hDPSCs was detected by MTT assays. (E∼H) The invasion of hDPSCs was detected by Transwell assays. (I, J) The formation of calcium nodules in hDPSCs was detected by Alizarin red staining. All experiments were conducted in triplicate (*p<0.05, **p<0.01, ***p< 0.001, vs. NC; ^p<0.05, ^^p<0.01, vs. siNC; #p<0.05, ##p<0.01, ###p<0.001, vs. MC; △p<0.05, △△p<0.01, vs. IC; ＋p< 0.05, ＋＋p<0.01, ＋＋＋p<0.001, vs. M; &p< 0.05, &&p<0.01, vs. Wnt1; §p<0.05, §§p< 0.01, vs. I; ‡p<0.05, ‡‡p<0.01, vs. siWnt1) (hDPSCs: human dental pulp stem cells, NC: negative control, siNC: small interfering RNA for negative control, MC: mimic control, IC: inhibitor control, M: miR-148a-3p mimic, I: miR-148a-3p inhibitor).

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  Fig. 6 MiR-148a-3p reversed the regulatory effect of Wnt1 on the activation of Wnt1/β-catenin and the expressions of DSPP, DMP-1, RUNX2, OCN, and Smad4. (A, B) The expressions of Wnt1, β-ca-tenin, DSPP, DMP-1, RUNX2, OCN, and Smad4 in hDPSCs were detected by RT-qPCR. GAPDH was used as an internal control. (C∼F) The protein expressions of Wnt1, β-catenin, DSPP, DMP-1, RUNX2, OCN, and Smad4 in hDPSCs were detected by Western blot. GAPDH was used as an internal control. All experiments were conducted in triplicate (***p<0.001, vs. NC; ^^^p< 0.001, vs. siNC; ###p<0.001, vs. MC; △△△p<0.001, vs. IC; ＋＋＋p<0.001, vs. M; &&&p<0.001, vs. Wnt1; §§§p< 0.001, vs. I; ‡‡‡p<0.001, vs. siWnt1) (hDPSCs: human dental pulp stem cells, NC: negative control, siNC: small interfering RNA for negative control, MC: mimic control, IC: inhibitor control, M: miR-148a-3p mimic, I: miR-148a-3p inhibitor).

Reference

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