METTL3 Promotes Osteogenic Differentiation of Human Periodontal Ligament Stem Cells through IGF2BP1-Mediated Regulation of Runx2 Stability.

N6-Methyladenosine (m6A) has been reported to play a dynamic role in osteoporosis and bone metabolism. However, whether m6A is involved in the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) remains unclear. Here, we found that methyltransferase-like 3 (METTL3) was up-regulated synchronously with m6A during the osteogenic differentiation of hPDLSCs. Functionally, lentivirus-mediated knockdown of METTL3 in hPDLSCs impaired osteogenic potential. Mechanistic analysis further showed that METTL3 knockdown decreased m6A methylation and reduced IGF2BP1-mediated stability of runt-related transcription factor 2 (Runx2) mRNA, which in turn inhibited osteogenic differentiation. Therefore, METTL3-based m6A modification favored osteogenic differentiation of hPDLSCs through IGF2BP1-mediated Runx2 mRNA stability. Our study shed light on the critical roles of m6A on regulation of osteogenic differentiation in hPDLSCs and served novel therapeutic approaches in vital periodontitis therapy.

RNA-sequencing analysis reveals the potential molecular mechanism of RAD54B in the proliferation of inflamed human dental pulp cells.

AIM: To investigate the role of RAD54B in the proliferation of inflamed human dental pulp cells (hDPCs) induced by lipopolysaccharide (LPS). METHODOLOGY: Normal, carious and pulpitic human dental pulp tissues were collected. Total RNA was subjected to RNA-sequencing (seq) and gene expression profiles were studied by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Differentially expressed genes (DEGs) in homologous recombination repair (HRR) were validated with qRT-PCR. The expression of RAD54B and TNF-α in human dental pulp tissues was detected using immunohistochemistry. HDPCs were cultured and RAD54B level in hDPCs was detected after LPS stimulation using western blot. CCK-8 was used to investigate the proliferation of hDPCs transfected with negative control (Nc) small interfering RNA (siRNA), RAD54B siRNA, P53 siRNA or both siRNAs with or without LPS stimulation. Flow cytometry was used to detect the cell cycle distribution, and western blot and immunofluorescence were used to analyse the expression of RAD54B, P53 and P21 under the above treatments. One-way and two-way anova followed by least significant difference posttest were used for statistical analysis. RESULTS: RNA-seq results identified DEGs amongst the three groups. KEGG pathway analysis revealed enrichment of DEGs in the replication and repair pathway. HRR and non-homologous end joining (NHEJ) components were further verified and qRT-PCR results were basically consistent with the sequencing data. RAD54B, an HRR accessory factor highly expressed in carious and pulpitic tissues as compared to that in normal pulps, was chosen as our gene of interest. High RAD54B expression was confirmed in inflamed human dental pulp tissues and LPS-stimulated hDPCs. Upon RAD54B knockdown, P53 and P21 expressions in hDPCs were upregulated whereas the proliferation was significantly downregulated, accompanied by increased G2/M phase arrest. After inhibiting P53 expression in RAD54B-knockdown hDPCs, P21 expression and cell proliferation were reversed. CONCLUSIONS: Gene expression profiles of normal, carious and pulpitic human dental pulp tissues were revealed. HRR components were elucidated to function in dental pulp inflammation. Amongst the DEGs in HRR, RAD54B regulated the proliferation of inflamed hDPCs via P53/P21 signalling. This research deepens our understanding of dental pulp inflammation and provides new insight to clarify the underlying mechanisms.

Identification of maternal serum biomarkers for prenatal diagnosis of nonsyndromic orofacial clefts.

Nonsyndromic orofacial clefts (NSOFCs) are the most common congenital defects in the oral and maxillofacial regions. It is mainly diagnosed prenatally through fetal ultrasonography. However, the accuracy of ultrasonography for NSOFC is unreliable. Maternal serological screening is a noninvasive method for the diagnosis of fetal malformations. In our study, we sought to identify specific biomarkers in maternal serum for predicting NSOFC prenatally. We quantified the alterations in maternal serum protein profiles between 20 pregnant women with NSOFC fetuses and 20 pregnant women with healthy fetuses by using isobaric tags for relative and absolute quantitation-based mass spectrometry (MS). The serum levels of 75 elevated and 50 decreased proteins in the NSOFC group were detected. Twenty-eight candidate biomarkers were selected for further confirmation by multiple reaction monitoring-MS; of these, 16 proteins were found to be significantly different. More importantly, the levels of three proteins (APOA, HPT, and CRP) were verified by ELISAs to be obviously altered in serum from pregnancies carrying fetuses with NSOFC. Our results indicate that analysis of the maternal serum proteome is a feasible strategy for biomarker discovery of NSOFC, and APOA, HPT, and CRP proteins are potential serum biomarkers for prenatal diagnosis of NSOFC.