Circular RNA CircFgfr2 Promotes Osteogenic Differentiation of Rat Dental Follicle Cells via Sponging MicroRNA miR-133a-3p and Up-regulating Distal-less Homeobox DLX3
Authors: Du Yu , Xu Cheng , Li Guixian , Li Jing , Jiang Hongwei , Ning Yang 
Published: January 2022

Abstract
BackgroundAs promising seed cells of periodontal tissue regeneration, dental follicle cells (DFCs) can differentiate into cementoblasts, osteoblasts and fibroblasts upon the regulation of signaling pathways. Emerging evidence shows that circular RNA (circRNA) may participate in tooth regeneration, however its effect on DFCs remains unclear. Our early research found that the expression of circFgfr2 was significantly up-regulated when the rat DFCs were incubated by mineral induction medium for 4 weeks, while the expression of miR-133 was down-regulated. It has been widely reported that circRNA may exert as a miRNA sponge to target genes. Therefore, the function of circFgfr2 in osteogenesis of rDFCs was further identified in this study.MethodsCircFgfr2 was identified by Sanger sequencing and agarose gel electrophoresis. The intracellular location of circFgfr2 in rDFCs was analyzed by fluorescence hybridization (FISH). Furthermore, we constructed circFgfr2 overexpressed rDFCs via pLC5-ciR plasmid. RNA high-throughput sequencing was carried out to investigate the differently expressed messenger RNA (mRNAs) between circFgfr2 overexpressed (OE) groups and control groups. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were performed to further explore the role of circFgfr2 in rDFCs. Moreover, the binding sites of miR-133a-3p and miR-133a-5p with circFgfr2 were predicted by bioinformatics and detected by dual-luciferase reporter (DLR) assay and Chromatin Isolation by RNA Purification (ChIRP). Bioinformatics analysis and the luciferase reporter assay were also performed to investigate the interaction between miR-133a-3p, miR-133a-5p and Distal-Less Homeobox 3 (DLX3). We also constructed miR-133a-3p overexpressed rDFCs via miR-133a-3p mimics. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to examine their expression of circFgfr2, miR-133a-3p, miR-133a-5p and bone-related genes including bone morphogenetic protein2 (BMP2), BMP6, Runt-related transcription factor 2 (RUNX2), osteocalcin (OCN), Osterix (OSX) and distal-less homeobox DLX3. Alkaline phosphatase (ALP) activity detection and staining along with Alizarin red staining and were performed to evaluate the mineralization ability of rDFCs. Western blot were performed to analyse RUNX2 protein expression.ResultsCircFgfr2 primarily had a cytoplasmic location. The mRNA sequencing results illustrated that 361 and 391 mRNAs were upregulated and downregulated respectively. GO and KEGG analysis indicated several pathways and processes which are important for osteogenic differentiation. Furthermore, circFgfr2 acted as a molecular sponge for miR-133a-3p which inhibited DLX3 expression. Overexpression of circFgfr2 increased expression levels of markers of osteogenesis including RUNX2, OCN, OSX, BMP2, BMP6 and DLX3 and decreased the expression of miR-133a-3p and miR-133a-5p. In addition, miR-133a-3p was shown to decrease mineralization in rDFCs. Moreover, circFgfr2 protected DLX3 and Runx2 from miR-133a-3p mediated suppression. ConclusionWe demonstrated that circFgfr2 promoted the osteogenic differentiation of rDFCs via circFgfr2 / miR-133a-3p / DLX3 / RUNX2 axis. CircFgfr2 has the potential to be the molecular target for the bone tissue engineering.