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

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

JOURNAL=Frontiers in Cell and Developmental Biology

VOLUME=9

YEAR=2021

URL=https://www.frontiersin.org/journals/cell-and-developmental-biology/articles/10.3389/fcell.2021.697243

DOI=10.3389/fcell.2021.697243

ISSN=2296-634X

ABSTRACT=<p>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 <italic>miRNA-221-3p</italic> in tooth development, through its loss or gain in function was evaluated. A variety of techniques were utilized to evaluate detailed functional roles of <italic>miRNA-221-3p</italic> during odontogenesis, including <italic>in vitro</italic> tooth cultivation, renal capsule transplantation, <italic>in situ</italic> hybridization, real-time PCR, and immunohistochemistry. Two-day <italic>in vitro</italic> tooth cultivation at E13 identified altered cellular events, including cellular proliferation, apoptosis, adhesion, and cytoskeletal arrangement, with the loss and gain of <italic>miRNA-221-3p</italic>. qPCR analysis revealed alterations in gene expression of tooth-related signaling molecules, including β<italic>-catenin</italic>, <italic>Bmp2</italic>, <italic>Bmp4</italic>, <italic>Fgf4</italic>, <italic>Ptch1</italic>, and <italic>Shh</italic>, when inhibited with <italic>miRNA-221-3p</italic> and mimic. Also, the inhibition of <italic>miRNA-221-3p</italic> demonstrated increased mesenchymal localizations of pSMAD1/5/8, alongside decreased expression patterns of <italic>Shh</italic> and <italic>Fgf4</italic> within inner enamel epithelium (IEE) in E13 + 2 days <italic>in vitro</italic> cultivated teeth. Moreover, 1-week renal transplantation of <italic>in vitro</italic> cultivated teeth had smaller tooth size with reduced enamel and dentin matrices, along with increased cellular proliferation and <italic>Shh</italic> expression along the Hertwig epithelial root sheath (HERS), within the inhibitor group. Similarly, in 3-week renal calcified teeth, the overexpression of <italic>miRNA-221-3p</italic> 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 <italic>miRNA-221-3p</italic> is required for the modulation of major signaling pathways, including Wnt, Bmp, and <italic>Shh</italic>, during tooth morphogenesis.</p>