Signaling Modulation by miRNA-221-3p During Tooth Morphogenesis in Mice.

Yam Prasad Aryal,Hitoshi Yamamoto,Sanggyu Lee,Tae-Young Kim,Ji-Youn Kim,Youngkyun Lee,Sanjiv Neupane,Chang-Hyeon An,Wern-Joo Sohn,Jae-Young Kim,Eui-Seon Lee

doi:10.3389/fcell.2021.697243

Abstract

miRNAs are conserved short non-coding RNAs that play a role in the modulation of various biological pathways during tissue and organ morphogenesis. In this study, the function of miRNA-221-3p in tooth development, through its loss or gain in function was evaluated. A variety of techniques were utilized to evaluate detailed functional roles of miRNA-221-3p during odontogenesis, including in vitro tooth cultivation, renal capsule transplantation, in situ hybridization, real-time PCR, and immunohistochemistry. Two-day in vitro tooth cultivation at E13 identified altered cellular events, including cellular proliferation, apoptosis, adhesion, and cytoskeletal arrangement, with the loss and gain of miRNA-221-3p. qPCR analysis revealed alterations in gene expression of tooth-related signaling molecules, including β-catenin, Bmp2, Bmp4, Fgf4, Ptch1, and Shh, when inhibited with miRNA-221-3p and mimic. Also, the inhibition of miRNA-221-3p demonstrated increased mesenchymal localizations of pSMAD1/5/8, alongside decreased expression patterns of Shh and Fgf4 within inner enamel epithelium (IEE) in E13 + 2 days in vitro cultivated teeth. Moreover, 1-week renal transplantation of in vitro cultivated teeth had smaller tooth size with reduced enamel and dentin matrices, along with increased cellular proliferation and Shh expression along the Hertwig epithelial root sheath (HERS), within the inhibitor group. Similarly, in 3-week renal calcified teeth, the overexpression of miRNA-221-3p did not affect tooth phenotype, while the loss of function resulted in long and slender teeth with short mesiodistal length. This study provides evidence that a suitable level of miRNA-221-3p is required for the modulation of major signaling pathways, including Wnt, Bmp, and Shh, during tooth morphogenesis.

Highlights

The well-defined stages in which tooth development progresses require an intricate and reciprocal signaling regulation between both the dental epithelium and neural-crest-derived mesenchyme (Balic and Thesleff, 2015)
The expression of miRNA-221-3p was restricted along the invaginated epithelium (Figure 1C) with intense expression noted along the enamel knot (EK), inner enamel epithelium (IEE), outer enamel epithelium (OEE), and dental papilla (DP) at cap stage (Figure 1D)
ROCK1, which is a downstream target of RhoA small GTP-binding proteins, regulates cell adhesion, cytokinesis, cell proliferation, and apoptosis and regulates actin cytoskeleton dynamics forming a complex with E-cadherin (Zohrabian et al, 2009; Smith et al, 2012)

Summary

Introduction

The well-defined stages in which tooth development progresses require an intricate and reciprocal signaling regulation between both the dental epithelium and neural-crest-derived mesenchyme (Balic and Thesleff, 2015). This reciprocal signaling frequently involves signaling via Bmp, Shh, Wnt, and Fgfs throughout the various stages of tooth development including initiation, bud, bell, cap, and root morphogenesis (Jussila and Thesleff, 2012). Functional studies including miRNA, siRNA, lncRNA, exosome, and so on have been utilized to define the developmental processes in tooth organogenesis (Jiang et al, 2017; Aryal et al, 2020; Huang et al, 2020) and require further research

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Conclusion